J. Parasitol., 95(2), 2009, pp. 424–433 ᭧ American Society of Parasitologists 2009 FOUR NEW SPECIES OF PLASMODIUM FROM NEW GUINEA LIZARDS: INTEGRATING MORPHOLOGY AND MOLECULES Susan L. Perkins and Christopher C. Austin* Sackler Institute for Comparative Genomics & Division of Invertebrate Zoology, American Museum of Natural History, Central Park West at 79th St., New York, New York 10024. e-mail: [email protected] ABSTRACT: New Guinea is one of the most biodiverse regions of the world, particularly in terms of the herpetofauna present, yet surprisingly little is known about the parasites that infect these organisms. A survey of diverse scinid and agamid lizard hosts from this country showed a diversity of malaria parasites infecting these hosts. We combined morphological and morphometric observations of the parasites (primarily gametocytes) along with DNA sequence data from the mitochondrial cytochrome b and cytochrome oxidase I genes and here describe 4 new species of Plasmodium, i.e. Plasmodium minuoviride n. sp., Plasmodium koreafense n. sp., Plasmodium megalotrypa n. sp., and Plasmodium gemini n. sp. A fifth species, Plasmodium lacertiliae Thomp- son and Hart 1946, is redescribed based on new observations of hosts and localities and additional molecular data. This combined morphological and molecular approach is advised for all future descriptions of new malaria parasite species, particularly in light of situations where every life-history stage is not available. Approximately 200 species of Plasmodium have been de- year. Thus, blood samples of vertebrates during typical field scribed from mammal, avian, and squamate reptile intermediate collections may reveal the presence of parasites, but often not hosts. These species descriptions have relied on classical meth- in all life stages. Still, the taxonomic description of these par- ods such as morphological characters, life-history traits, and asites as species is valuable as a means of quantifying biodi- host taxon, all of which are useful, but all of which are also versity and providing a framework within which additional subject to serious shortcomings. For example, appearance of the study, including more detailed studies of the life cycle, can be parasites on thin blood films can be altered by the rate of drying performed. Until recently, however, a means with which to link of the original material and staining protocol. Also, character- samples from multiple hosts taken at different times has not istics long held as definitive for the classification of parasites been possible. into genera and subgenera, including the production of pigment The use of DNA sequences has recently started to serve as and the presence of erythrocytic schizonts, are now thought to a powerful bridge to allow for the diagnosis of species when be considerably more plastic. Examples of this include the they are present in different life-history stages, i.e., the eggs of monophyly of parasites such as Plasmodium azurophilum Tel- fish (Desalle and Birstein, 1996), larval and adult arthropods ford, 1975, a lizard parasite that does not produce pigment, and (Barber and Boyce, 2006; Ahrens et al., 2007; Zhou et al., Hepatocystis species, mammalian parasites that do not have 2007), and efficient diagnosis and study of parasites with com- erythrocytic schizonts, with other species of Plasmodium, plex life cycles (Brant et al., 2006). The iterative process of which is defined with the characters of pigment production and sequencing a gene or genes from parasites, the phylogenetic erythrocytic schizogony (Perkins and Schall, 2002; Martinsen study of those sequences in the context of other detected infec- et al., 2006, 2008). Ideally, to describe a species of Plasmodi- tions and samples of previously described species, and then the um, data provided would include morphology of all stages, in deposition of these sequences into databases can provide an both the vertebrate and insect hosts, from multiple infections, efficient and cohesive process for the taxonomic study of par- as well as information on the complete life cycle (timing of all asites. DNA sequences should always be coupled with morpho- developmental stages), and the full range of host and vector logical observations of at least some of the life-history stages species. Very few Plasmodium species have all of this kind of so that independent lines of evidence are available for species information available, and those that do are primarily those spe- identity and description (DeSalle et al., 2005). cies that infect humans or the mammalian parasites that are In this study we conducted a large survey of the lizards, model organisms. In many cases, only a few—and sometimes primarily skinks, from several sites in one of the most biolog- just a single—blood smears have been available when defining ically diverse regions of the world, Papua New Guinea, taking new species; this is clearly far from the ideal method. blood smears and tissue collections from each. New Guinea has For various reasons, sometimes the acquisition and study of been recognized as one of the world’s 5 ‘High Biodiversity all parasite life stages, or even all of those typically used for Wilderness Areas,’ and, as the world’s largest and tallest trop- species descriptions of Plasmodium species, are not possible. ical island, it represents the third largest tract of intact rainforest Many malaria parasites, from those infecting humans (e.g., Ha- after the Amazon and Congo basins (Austin, 1995; Mittermeier mad et al., 2002; Drakeley et al., 2006) to those infecting birds et al., 2003). The island occupies less than 0.6% of global land (Cosgrove et al., 2008) and lizards (Goodwin and Stapleton, area, but it is estimated to harbor 5–7% of the world’s biodi- 1952; Jordan and Friend, 1971; Bromwich and Schall, 1986), versity (Beehler, 1993; Dinerstein and Wikramanayake, 1993; show marked seasonality, where there is an almost complete Mack, 1998; Myers et al., 2000). Much of this estimated di- absence of various life-history stages at certain points of the versity is not known to science. In particular, the parasites of New Guinea wildlife, including hemoparasites, are poorly stud- Received 19 June 2008; revised 6 August 2008; accepted 18 Septem- ied (Austin and Perkins, 2006; Klompen and Austin, 2007). In ber 2008. the course of this survey, we encountered several parasites that * Department of Biological Sciences & Museum of Natural Science, Louisiana State University, 119 Foster Hall, Baton Rouge, Louisiana were morphologically distinct. However, in each case, very few 70803. asexual stages were present; thus, classifying the parasites, even 424 PERKINS AND AUSTIN—NEW SPECIES OF PLASMODIUM IN NEW GUINEA LIZARDS 425 at the generic level, would be problematic if only morpholog- quences were edited in Sequencher (Gene Codes, Madison, Wisconsin). ical characters were used. Therefore, we also used sequences There was no length variation in the sequences, and alignments were done simply by eye. Several published sequences of other lizard malaria from 2 genes and a phylogenetic analysis to determine that parasite species (Martinsen et al., 2008) were added to the matrix of these parasites are indeed Plasmodium, and that they are dis- the newly generated sequences and the mammal-infecting taxa; Plas- tinct enough, particularly when combined with morphological modium berghei and Plasmodium vivax were used as outgroups. The 2 characteristics of the gametocyte stages, to define as new spe- genes were combined, and primer sequences were removed before anal- ysis. Phylogenetic analyses were conducted using maximum likelihood cies. The combination of morphological data and morphomet- using RaxML (Stamatakis, 2006) via the Cyberinfrastructure for Phy- rics of gametocytes with sequences of the parasites’ cytochrome logenetic Research (CIPRES) portal (http://www.phylo.org/news/ b(cytb) and cytochrome oxidase I (coxI) genes from the mi- RAxML), and the data set was partitioned by gene such that separate tochondrial genome shows 5 monophyletic lineages, 4 of which models of evolution were estimated for each. Bootstrapping (cut-off appear to be new to science (3 from scincid lizard hosts and 1 automatically determined by the software) was used to assess nodal support. from an agamid lizard), and a fifth species, which we conser- Molecular characters for each of the 4 new species and the redescrip- vatively place within Plasmodium lacertiliae, originally report- tion of Plasmodium lacertiliae were determined by aligning sequences ed on Goodenough Island, off the coast of the main island of to those published for named species of lizard and bird malaria para- New Guinea (Thompson and Hart, 1946). sites. These included Plasmodium mexicanum (NC࿞009960), Plasmo- dium floridense (NC࿞009961), Plasmodium gallinaceum (EU254535 and EU254578), Plasmodium relictum (AY733090), Plasmodium azur- MATERIALS AND METHODS ophilum (in erythrocytes; EU254532 and EU254575), Plasmodium Lizards were captured alive by hand or blowpipe. Blood was col- azurophilum (in leucocytes; EU254533 and EU254576), Plasmodium lected via standard protocols with a heparinized capillary tube from the giganteum (EU254534 and EU254577), Plasmodium chiricahuae (cytb post-orbital sinus. Thin blood smears on slides were air-dried and then only; AY099061), Plasmodium elongatum (cytb only; AF069611), Plas- fixed in 95% methanol. All thin blood smears are currently held at the modium agamae (cytb only; AY099048), Haemoproteus kopki (cytb American Museum of Natural