ARTICLE IN PRESS Protist, Vol. 157, 173—183, April 2006 http://www.elsevier.de/protis Published online date 11 April 2006 ORIGINAL PAPER Phylogenetic Analysis of Coccidian Parasites from Invertebrates: Search for Missing Links Jana Kopecˇ na´ a,b, Milan Jirku˚ a,b, Miroslav Obornı´ka,b, Youri S. Tokarevc, Julius Lukesˇ a,b, and David Modry´ a,d,1 aInstitute of Parasitology, Czech Academy of Sciences, Cˇ eske´ Budeˇ jovice, Czech Republic bFaculty of Biology, University of South Bohemia, Cˇ eske´ Budeˇ jovice, Czech Republic cAll-Russian Institute for Plant Protection, St. Petersburg, Russian Federation dDepartment of Parasitology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic Submitted September 23, 2005; Accepted February 13, 2006 Monitoring Editor: Frank C. Seeber Apicomplexan parasites represent one of the most important groups of parasitic unicellular eukaryotes comprising such important human parasites such as Plasmodium spp. and Toxoplasma gondii. Apicomplexan radiation as well as their adaptation to the parasitic style of life took place before the era of vertebrates. Thus, invertebrates were the first hosts of apicomplexan parasites that switched to vertebrates later in evolution. Despite this fact, apicomplexan parasites of invertebrates, with the exception of gregarines, have so far been ignored in phylogenetic studies. To address this issue, we sequenced the nuclear SSU rRNA genes from the homoxenous apicomplexan parasites of insects Adelina grylli and Adelina dimidiata, and the heteroxenous Aggregata octopiana and Aggregata eberthii that are transmitted between cephalopods and crustaceans, and used them for phylogenetic reconstructions. The position of the adelinids as a sister group to Hepatozoon spp. within the suborder Adeleorina was stable regardless of the phylogenetic method used. In contrast, both members of the genus Aggregata possess highly divergent SSU rRNA genes with an unusual nucleotide composition. Because of this, they form the longest branches in the tree and their position is variable. However, the genus Aggregata branches together with adelinids and hepatozoons in most of the analyses, although their position within the scope of this cluster is unstable. & 2006 Elsevier GmbH. All rights reserved. Key words: Apicomplexa; Eucoccidiorida; Adelina; Aggregata; molecular phylogeny; SSU rDNA. Introduction The phylum Apicomplexa comprises important members of this phylum is important not only human parasites, such as Plasmodium spp., the from an evolutionary point of view, but also for causative agents of malaria, and Toxoplasma proper diagnosis and treatment of infections gondii, which is widely dispersed even in human caused by Apicomplexa. One of the main populations of developed countries. Therefore, problems influencing the accuracy of current our understanding of the relationships among apicomplexan phylogenetic analyses is an inap- propriate taxon sampling. While some apicom- 1 Corresponding author; plexan groups, such as coccidians from fax 42 541562266 vertebrate hosts, are well represented in data sets e-mail [email protected] (D. Modry´ ). for phylogenetic reconstructions, their relatives & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2006.02.005 ARTICLE IN PRESS 174 J. Kopecˇ na´ et al. parasitizing invertebrates have so far been largely The genus Aggregata Frenzel, 1885 was estab- ignored (Barta 2001; Jirku˚ et al. 2002; Morrison et lished 150 years ago to embrace digenetic al. 2004; Sˇ lapeta et al. 2003). parasites that are transmitted between cephalo- Apicomplexans are intracellular, largely non- pods and crustaceans (Dobell 1925). Merogonial flagellated organisms that are classified together development occurs in the intestine of crabs and with ciliates and dinoflagellates within the Alveo- shrimps and proceeds by gamogony and spor- lata. Apicomplexans are purely parasitic organ- ogony upon ingestion by a squid or octopus isms. Therefore, the transition from the predation (Gestal et al. 2002; Porchet-Hennere´ et al. 1981). to intracellular parasitism is probably the most The octopuses in Naples were so heavily infected striking feature in their evolution. It has been that Dobell (1925) complained about the impossi- suggested that this event occurred approximately bility to experimentally complete the life cycles at the time of the divergence of dinoflagellates due to the lack of uninfected definitive hosts. Ever and apicomplexans (Kuvardina et al. 2002; since, various Aggregata species have been Leander and Keeling 2003, 2004). The time of reported as a very frequent parasite of different further radiation of the phylum Apicomplexa is cephalopods in the Atlantic Ocean, the North Sea, placed in the Precambrian period, 800 Mya the Mediterranean, and other places (Gestal et al. (Escalante and Ayala 1995), when multicellular 2002; Porchet-Hennere´ et al. 1981; Sardella and organisms are supposed to have emerged. It Martonelli 1997). The family Aggregatidae Labbe´ , clearly predates the appearance of vertebrates grouping Aggregata spp. together with the genera and most likely that of invertebrates as well Merocystis Dakin, 1911 and Selysia Dubosq, (Lynch 1999). Since all currently known apicom- 1917, is traditionally classified within the suborder plexans are obligatory parasites, it is reasonable Eimeriorina (Dobell 1925; Gestal et al. 2005; Upton to assume that the ancestral lineages initially 2000). evolved in the invertebrate hosts, probably even In a first attempt to fill the gaps in our knowl- before the Cambrian explosion. It is generally edge about the phylogenetic position of cocci- accepted that the earliest apicomplexans are dians from invertebrates, we herein present results archigregarines (Leander et al. 2003). However, of phylogenetic analyses of members of the further evolution of main lineages within the order genera Adelina and Aggregata parasitizing arthro- Eucoccidiorida is unclear. Any quest for the pods and molluscs. ancestral forms of the main lineages should therefore be focused on the apicomplexans parasitizing terrestrial and aquatic invertebrate Results hosts. The absence of molecular data for any representative (!) of Eucoccidiorida from this group Two adelinid species that differ in their endogen- of hosts in phylogenetic analyses is alarming, as it ous development were selected: Adelina dimidiata may distort or at least conceal the correct Schneider, 1875, a typical intestinal parasite of relationships between the major apicomplexan insects (Fig. 1), and Ad. grylli Butaeva, 1996 with groups. Interestingly, vector-transmitted apicom- an extraintestinally located endogenous develop- plexans (Haemospororina, Adeleorina and Piro- ment (Fig. 2)(Sokolova et al. 1999). Furthermore, plasmorina) retain the sexual part of their life cycle two representatives of the genus Aggregata, in the invertebrate hosts, which suggests that namely Aggregata octopiana and Agg. eberthii, these heteroxenous parasites have evolved from both from Mediterranean cephalopods were also monoxenous apicomplexans of invertebrates included in this study. These coccidians are (Barta 1989). Members of the coccidian genera characterized by the formation of large, macro- Adelina and Aggregata, so far considered unre- scopically visible oocysts in the viscera of cepha- lated (Barta 2000; Upton 2000), have been lopods (Fig. 3), each containing hundreds of isolated from ecologically distant invertebrates, polyzoic sporocysts (Figs 4, 5). To establish the and used in this study. The genus Adelina Hesse, phylogenetic position of the above-mentioned 1911 comprises monoxenous apicomplexans species, we have sequenced their entire SSU usually parasitizing insects. Based on the pre- rRNA genes. When compared to other available sence of syzygy in their life cycle, adelinids are homologs, sequences of the genus Adelina were traditionally referred to as members of the order not exceptionally divergent, and their phylogenetic Adeleorina, which also comprises heteroxenous position was very stable in all constructed trees haemogregarines alternating between vertebrate (Figs 7, 8, and data not shown). In contrast, the and invertebrate hosts (Barta 2000). SSU rRNA genes of both Aggregata species had ARTICLE IN PRESS Phylogeny of Coccidian Parasites from Invertebrates 175 Figures 1—5. Representative morphology of oocysts of the studied taxa. 1. Exogenously sporulated oocyst of Adelina dimidiata from the feces of Scolopendra cingulata (Bar ¼ 30 mm). 2. In situ sporulated oocysts of Adelina grylli from the fat body of Gryllus bimaculatus (Bar ¼ 20 mm). 3. Macroscopic oocysts of Aggregata octopiana on the surface of dissected intestine of Octopus vulgaris (Bar ¼ 1 cm). 4. Micrograph of a single oocyst of Aggregata octopiana containing thousands of in situ sporulated sporocysts (Bar ¼ 200 mm). 5. Detail of sporocysts enclosing spirally coiled elongated sporozoites in a head-to-tail position (Bar ¼ 20 mm). an unbalanced nucleotide composition, shifted that the studied SSU rRNA gene is derived from towards a very low cytosine content. As shown in the oocysts of Aggregata (not shown). Figure 6, the SSU rRNA genes of Aggregata spp. For phylogenetic analysis, three different align- contain less than a half of cytosines compared to ments were constructed. Alignment I (1284 nu- the same genes in other coccidia. The nucleotide cleotides, see Methods for details) was used to Blast search identified a number of relatively high specify the position of the genera Adelina and matches from outside