doi 10.1098/rspb.2000.1290 Speciesconceptsandmalariaparasites: detecting acrypticspeciesof Plasmodium Susan L.Perki ns { Department of Biology,University of Vermont, Burlington,VT 05405,USA Species ofmalaria parasite (phylum Apicomplexa: genus Plasmodium)havetraditionally been described usingthe similarity species concept(based primarily on di¡ erences inmorphological or life-history characteristics).Thebiological species concept(reproductive isolation) and phylogenetic species concept (basedon monophyly) have not been used beforein de¢ ning species of Plasmodium. Plasmodium azurophilum ,described from Anolis lizardsin the eastern Caribbean,is actuallya two-species cryptic complex.The parasites werestudied from eightislands, from Puerto Rico in the northto Grenada in the south.Morphology of the twospecies isverysimilar (di¡erences areindistinguishable to the eye),but one infects onlyerythrocytes andthe otheronly white blood cells. Moleculardata for the cytochrome b gene revealthat the twoforms arereproductively isolated ;distinct haplotypesare present oneachisland and arenever shared between the erythrocyte-infectingand leucocyte-infecting species. Eachforms amono- phyleticlineage indicating that theydiverged before becoming established inthe anolesof the eastern Caribbean.This comparison of the similarity,biologicaland phylogenetic species concepts formalaria parasites revealsthe limited valueof usingonly similarity measures inde¢ ning protozoan species. Keywords: Plasmodium;species concepts; cryptic species; malaria fora givenspecies ofparasite ( Buckling et al. 1997; 1.INTRODUCTION Escalante et al.1998;Eisen &Schall2000) . In1 842,Charles Darwin sketched outa lengthyoutline of Thebiological species concept( BSC)de¢ nes species as whatwould become the Originof species ,then set it aside `groupsof actively or potentially interbreeding popu- formany years while he assembled the necessary back- lationswhich are reproductively isolated from other such groundinformation in support of his majorhypotheses. populations’(Mayr 1963) .Althoughit haslong been Primaryto this e¡ort wasthe needto clarify the concept popularamong taxonomists of metazoan organisms, the ofspecies (Darwin1 958).Acenturyand a halflater ,this BSCis inadequatefor many parasites including goalstill presents avexingproblem in modern biology Plasmodium becauseit uses criteria withsevere limitations andgenerates substantial controversy .Thenumber of forasexually reproducing organisms. Although di¡erent species concepts continuesto grow ;indeed Plasmodium doeshave a sexuallyreproductive stage, Mayden(1 997)catalogued 22 species concepts that are almostno explicit data are available on reproductive currently inuse. Thesede¢ nitions can be grouped into isolationamong de¢ ned species. Infact, a debate three broadclasses: similarity species concepts that are continuesover the degreeof clonality in these protozoan basedon unique phenotypic features ofthe organisms; parasites (Dye 1991;Tibayrenc& Ayala1 991;Walliker biologicalor reproductive species concepts that centre on 1991; Paul et al.1995;De Meeuªs et al.1998).Finally,the the abilityof sexually reproducing organisms to cross and degreeto which populations of parasites mayoverlap and producefertile o¡spring ;andphylogenetic or lineage- potentiallyinterbreed canlargely be a functionof their basedconcepts that emphasize sharedevolutionary hosts’ distribution,both past andpresent (Killick- historyof populations (monophyly) .Ideally,descriptions Kendrick1 978),andnot a limitationof their own ofspecies shouldaddress eachof these three maintypes reproductivecompatibilities. ofconcepts, but inpractice this isoftennot feasible. Theadvent of molecular genetic techniques such as Themalaria parasites (phylumApicomplexa: genus DNA sequencinghas allowed a novelinsight into the de¢- Plasmodium),present acase studyin the di¤culties of nitionof species. Cracraft( 1983)de¢ ned species undera de¢ning species. Until recently, Plasmodium species were lineage-basedperspective, termed the phylogenetic described basedentirely on phenotypic similarity species concept,as `the smallest diagnosablecluster of measures includingmorphology of the stages infecting individualorganisms within which there is apattern of vertebrate bloodcells, life-historytraits, species ofhost ancestryand descent’ .Thephylogenetic species concept infected orsymptoms ofinfection(Garnham 1 966).These hasbeen used todescribe numerousinstances ofcryptic maybe unreliable characteristics, however.F orexample, species complexes,groups of organisms that areindistin- the parasitecells seen underthe microscopehave been guishablemorphologically ,yetshow as much genetic severelydistorted inappearance by preservation and di¡erentiation as described species. Examplesinclude staining,parasites mayinfect awiderange of host species, well-studied insect systems such as Anopheles mosquitoes andlife-history traits varysubstantially among infections (White 1974;Lounibos et al.1998),other invertebratetaxa (Sturmbauer et al.1999),reptiles (Bruna et al. 1996) and evenlarge birds (Baker et al.1995).Manyparasite species {Present address:Division of Invertebrates, American Museum of NaturalHistory ,CentralPark W estat 79th St, NewY ork,NY 10024, havealso been found to comprise species complexes(e.g. USA ([email protected]). Lymbery& Thompson1 996;Hung et al.1999).Molecular Proc. R.Soc.Lond. B (2000) 267, 2345^2350 2345 © 2000The RoyalSociety Received 18April 2000 Accepted 4August 2000 2346S. L. Perkins Species concepts and malariaparasites parasites must quicklycycle from one cell class toanother duringan infection. Here Ishowusing DNA sequencingof P.azurophilum sensu Telfordthat these parasites arein fact composed of twogenetically distinct lineages öonethat infects erythrocytes andone that infects whiteblood cells. Althoughthe twoparasites areindistinguishable morpho- logicallyunder the lightmicroscope (except forthe obviousdistinction of host cell class),theyare repro- ductivelyisolated and have had independent evolutionary histories inthe Caribbean.This appears to be the ¢rst such comparativedata for any malaria parasite. 2.MATERIAL AND METHODS (a) Parasite sampling P.azurophilum isfound throughout the eastern Caribbean, Figure 1. P.azurophilumsensu Telford(1975) in lizardblood fromPuerto Rico in thenorth to Grenada in thesouth (Staats cells. (a)Gametocytein erythrocyte.( b)Schizontin &Schall1996 a;thisstudy) .Infectedlizards were collected from erythrocyte.( c)Gametocytein leucocyte.( d ) Schizont eightislands (Puerto Rico, Saba, St Kitts, Guadeloupe, (andgametocyte) in leucocyte. Dominica,Martinique, St Vincent and Grenada) either by hand orby slip noose. A toeclip was used to obtain blood both for a thinsmear and to blot ¢ lterpaper for subsequent DNA dataare beginning to help uncover the relationshipsfor extraction.All lizardswere returned to their place of capture some species of Plasmodium (primarilythose infecting within 24hasper the protocol approved by the U niversityof mammals),but such dataon intraspeci¢ c relationships VermontAnimal Care and Use Committee. Thin smearswere arescant. Exceptionsto this arethe classic studies by stainedwith Giemsaand scanned for six or more minutes at Wallikerand others (summarized inBeale & Walliker 1000.Only those infections in which parasiteswere observed £ 1988)on enzymaticdi¡ erences ofvarious rodent malaria exclusivelyin onecell class were used for any further genetic subspecies andrecent workconcerning the human work;however,mixed infections were used for morphological malariaparasite, Plasmodium vivax (Qari et al.1993;Esca- measurementssince each cell was measured independently and lante et al.1995).Here Ipresent evidencefor one such classi¢ed byhost cell type (erythrocyte or leucocyte) . instanceof a cryptic species of Plasmodium and support myconclusions with data relevant for de¢ ning these para- (b) Morphometric tests sites basedon similarity ,biological(reproductive) and Parasiteswere measured from 1 2naturalinfections from phylogeneticspecies concepts. Anolisgundlachi fromthe El V erdeField Station, Puerto Rico. Plasmodium azurophilum is amalariaparasite of Anolis Eachblood smear was scanned at 1000and parasites were £ lizardsin the Caribbeanislands. In his species descrip- chosenfor measurement if judgedto be of mature morphology tion,T elford( 1975)noted that these parasites appearedto (largestsize for both schizonts and gametocytes, largest number undergoboth asexual replication (schizogony) and ofmerozoites for schizonts and distinct staining for male and productionof sexual stages orgametocytes(gamegony) in femalegametocytes) .Parasitecells were measured using a botherythrocytes andseveral classes ofwhite blood cells drawingtube and a ZIDAS(Zeiss, Thornwood, NY ,USA) (¢gure 1 ).Thiswas unusual because at the time onlyone imageanalysis system. Three size measurements were taken for other Plasmodium species (Plasmodium mexicanum ) had been eachparasite: (i) parasite cell area in squaremicrometres; (ii) notedto exploit cells other thanerythrocytes ofthe verte- parasitemaximum diameter in micrometres;and (iii) parasite bratehost, and then onlyin exceedingly heavy infections minimumdiameter in micrometres.Three measures of parasite (Ayala1 970;J ordan1970) .Telford( 1975)found no cellshape were also taken: (i) parasite minimum diameter / morphologicaldi¡ erences forparasites inerythrocytes maximumdiameter ;(ii)parasite circle diameter (the area of an versus leucocytes,and data from course- of-infection irregularshape transformed