Species Concepts and Malaria Parasites

Home , Apicomplexa, Plasmodium azurophilum

doi 10.1098/rspb.2000.1290

Speciesconceptsandmalariaparasites: detecting acrypticspeciesof Plasmodium Susan L.Perkins { Department of Biology,University of Vermont, Burlington,VT 05405,USA Species ofmalaria parasite (phylum Apicomplexa: genus Plasmodium)havetraditionally been described usingthe similarityspecies 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 fromeight islands, from Puerto Rico in the northto Grenada in the south.Morphology of the twospecies isverysimilar (di¡erences areindistinguishable to the eye),butone 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: similarityspecies 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 thatemphasize shared evolutionary 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, butin practice 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

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 reproductivelyisolated 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),butsuch 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 into an equivalentcircle area); and studies suggestedthat the parasites mightbe switching (iii)parasite form factor ((4 ºarea)/perimeter2). fromone host cell class toanother during their lifecycle. Afewyears later, A yala& Hertz (1981)reported (c) DNAextraction, ampli¢cation andsequencing P.azurophilum in Anolis roquet lizardsfrom Martinique, yet Foreach island, six infections were chosen for genetic notedthat the forms inerythrocytes andthose inleuco- analysis,three consisting of parasites only in erythrocytesand cytes werenever observed in the same lizard,nor even at threewith parasitesonly in whiteblood cells, with twoexcep- the same collectingsite, andthey concluded that, `were it tions.On Grenada, infections were found only in erythrocytes, notfor T elford’sthorough description, our plasmodia andon Guadeloupe just one lizard was found to be infectedwith wouldunhesitatingly have been identi¢ ed as two distinct P.azurophilum in erythrocytesonly and just one lizard was species’.Ina subsequent studyof P.azurophilum from St infectedwith P.azurophilum in whiteblood cells only .DNAwas Martin island,Schall ( 1992)observed sites andeven extractedfrom blood dried on the ¢ lterpaper using sodium occasionallyindividual lizards with both forms, yetnoted dodecylsulphate lysis followed either by phenol ^chloroform that most P.azurophilum infectionswere exclusively either extraction,and ethanol precipitation and washing, or byammo- inerythrocytes orin leucocytes and proposed that the niumacetate (7 .5M) precipitationof proteins with subsequent

Proc. R.Soc.Lond. B (2000) Species concepts and malariaparasites S.L. Perkins2347

Dominica red maximum-likelihoodanalyses, a setof 40 evolutionary models 0.005 changes wastested on the neighbour-joining tree obtained from an 91 Saba, Guadeloupe red initialsearch using the program Model Test( Posada& Crandall 70 St Kitts red (a) 1998).The simplest,best model (via log-likelihood ratio tests) 74 St Vincent, Martinique red wasthen chosen for the maximum-likelihood algorithm. This modelconsisted of a six-step(general time reversible) matrix, 92 Grenada red proportionof invariable sites 0andgamma shape parameter ˆ 74 St Kitts red (b) of0.0102. The Kishino ^Hasegawatest (K ^Htest;Kishino & Puerto Rico red Hasegawa1989) was used as a statisticaltest of alternativereso- lutionsof thephylogeny .Forthis test, two competing hypotheses Dominica white werecompared: (i) P.azurophilum inerythrocytes and 90 Saba, Guadeloupe white P.azurophilum in whiteblood cells each form monophyletic groups;versus (ii) the erythrocytic forms and white blood cell- Puerto Rico white 98 infectingforms from each island are each other’s closest relatives. St Kitts white 78 St Vincent, Martinique white 3. RESULTS Plasmodium gallinaceum (a) Morphometric analyses Adiscriminate analysiswas used todetermine the use Figure2. Neighbour-join ingtree of P.azurophilum haplotypes, ofmorphological measurements indi¡ erentiating rootedwith theavian malaria parasite, P.gallinaceum . Red, P.azurophilum cells inerythrocytes versus leucocytes. infectionin erythrocytes;white, infection in leukocytes.Node Discriminate scores successfully classi¢ed 46 out of 64 supportwas determined with 1000replicates of jackkni¢ng (72%)of gametocytes (likelihood ratio test, p 0.0005), with 33%deletion in each. ˆ but failedto distinguish schizonts basedon cell type exploited(50 out of 82, 61% successfully classi¢ed, isopropanolprecipitation and ethanol washing. A fragmentof p 4 0.05).When onlymeasurements relatedto parasite themitochondrial cytochrome b genewas ampli¢ ed using size (parasitearea, maximum diameter ,minimum primersDW 1(5 ’-TCAACAATGACTTTATTTGG-3 ’) and diameter) wereused, the discriminate analysisagain DW3(5’-TGCTGTATCATACCCTAAAG-3 ’),which producea successfully classi¢ed 4 1outof 64 (64%) of the cells 673bp product (Creasey et al.1993).Reactionswere set up in (p 0.031)forgametocytes, but wasnot reliable for ˆ 25 m lPCRswith Ready-to-Go TM PCR beads( Pharmacia schizonts (45out of 82 or 55%, p 5 0.05).Usingthe Biotech,Piscataway ,NJ,USA) using 1.5 mM MgCl 2, 2.5 m M of shapeparameters alonedid not allow discrimination of eachprimer and ca.250ng of genomic DNA. Ampli¢ cation either gametocytes(52%, p 4 0.05)or schizonts (57%, reactionswere subjected to 35 cycles of 94 8Cfor1 min,48 8C for p 4 0.05).Althoughthe compositesize measure was 1min, and 72 8Cfor1 minand then 72 8Cfor5 min.F orweak successful indistinguishing gametocytes in the twohost infections,a nestedPCR designwas employed using outer cell classes, eachmeasure alonedid not di¡ er forparasites primersDW2 (5 ’-TAATGCCTAGACGTATTCCTGATTATC inhost erythrocytes versus leucocytes( U-tests, p 4 0.05). CAG-3’,self-designed)and DW4 (5 ’-TGTTTGCTTGGGAGC TGTAATCATAATGTG-3 ’ AL1356;Escalante et al. 1998) and (b) Phylogenetic analyses ˆ subjectedto 35 cycles of 94 8Cfor1 min,6 0 8Cfor1 minand Atotalof 1 2haplotypesof this fragmentof the 72 8Cfor1 minwith a¢nalpolymerization step of 5 minat cytochrome b genewere identi¢ ed, ranging from 0. 18to 72 8C. A0.5l aliquotof this product was used as a templatefor 3.1%in sequence divergence.All samples sequencedfor a anestedreaction with primersDW 1andDW3 under the same givencell class wereidentical on each island with the conditionsas above. PCR productswere puri¢ ed with Nanosep exceptionof St Kitts wheretwo haplotypes of 100Kcolumns ( PallGelman, Ann Arbor,MI, USA),subjected P.azurophilum inerythrocytes wereobserved. Several tocycle sequencing using both forward and reverse primers islandsshared haplotypes: Saba and Guadeloupe for (DW1andDW3) and run on an ABI (Applied Biosystems, P.azurophilum inerythrocytes, Martiniqueand St Vincent FosterCity ,CA, USA)Prism automated sequencer (V ermont inerythrocytes, Sabaand Guadeloupe in leucocytes, and CancerCenter, University of Vermont). Martiniqueand St Vincent inleucocytes. These were pooledfor all phylogenetic analyses. The alignment of (d) Phylogenetic analysis eachof these haplotypesshowed four ¢ xeddi¡ erences Atotalof 554 bp of the cytochrome b genewere used for this betweenthe parasites inerythrocytes andthose infecting analysis (5’ nucleotideswere removed so that the sequences leucocytesand no haplotypes were shared between couldbe aligned with thepublished sequence for Plasmodium infectionsin red andwhite blood cells. Itis possiblethat gallinaceum ,anavian malaria parasite that was used as the this assortment ofhaplotypes was simply a result of outgroup,GenBank accession numbers AF06961 2).All phylo- randomsampling. However, the maximumprobability of geneticanalyses were done using P AUP * 4.0b4(Swo¡ ord 1 999). this occurringmay be calculatedas follows.If weassume Threemethods of tree construction were employed: neighbour that the twohaplotypes occur in a 1:1ratio(the joining,unweighted maximum parsimony ,andmaximum likeli- probabilitywould be lowerif other ratios occur) ,then for hood.Neighbour-joining trees were calculated using T amura ^ eachisland with three infectionsin each cell class Neidistances. These were appropriate due to the high A-T bias sequenced,the probabilitywould be ( 1 0.5 0.5) £ £ (average 3.5%)for these sequences (T amura& Nei1 984).For (0.5 0.5 0.5) 0.03,and the totalprobability would ˆ £ £ £ ˆ

Proc. R.Soc.Lond. B (2000) 2348S. L. Perkins Species conceptsand malariaparasites

Dominica red Dominica red 0.005 substitutions per site 88 Saba, Guadeloupe red Saba, Guadeloupe red St Kitts red (a) St Kitts red (a) Grenada red 82 84 St Kitts red (b) St Vincent, Martinique red Puerto Rico red St Kitts red (b) Grenada red Puerto Rico red St Vincent, Martinique red Dominica white Dominica white Saba, Guadeloupe white 89 Saba, Guadeloupe white Puerto Rico white St Kitts white 97 Puerto Rico white St Vincent, Martinique white St Kitts white 73 Plasmodium gallinaceum St Vincent, Martinique white Figure4. Maximum-likeli hoodphylogeny of P.azurophilum haplotypesconstructed with ageneraltime reversible Plasmodium gallinaceum (six-parameter)model of substitution,proportion of invariable sites 0anda gammashape parameter of 0.0102,rooted Figure3. Strict consensus of fourequally parsimonious ˆ with theavian malaria parasite, P.gallinaceum .Red,infection trees,rooted with theavian malaria parasite, P.gallinaceum . Red,infection in erythrocytes;white,infection in leucocytes. inerythrocytes;white, infection in leucocytes.The log-likelihoodscore of thephylogeny is 7 1044.15. The consistencyindex is 0.893 and the retention index is 0.878.The lengthof the tree is 56steps. Node support wasdetermined with 1000replicates of jackkni¢ng with 33% microscope.The morphology and life history of parasites deletionof characteristicsin each. mayalso vary according to the host species inwhich they ¢ndthemselves (Manwell1 943;Jordan 1 975).Finally,a bethe productover all islands. Thus, the probabilityof phylogeneticstudy of Plasmodium andother genera of apparentdivision of haplotypes by cell class occurringby malariaparasites (S. L. Perkinsand J .J.Schall,unpub- chanceduring the samplingwas 5. 7 1078. lisheddata) has shown that bothlife-history traits and £ Allthree methods oftree constructionrevealed two host species infected areuninformative for revealing distinct monophyleticgroups of parasites: oneinfecting evolutionaryrelationships among taxa. The practice of erythrocytes andone infecting white blood cells (¢gures usingsimilarity-based species concepts hasbeen very 2^4),eachwith strong nodal support for this division. useful,but conceivably has led to the splittingof true Themaximum-parsimony analysis produced four trees; a species basedon inter-infection variationas well as the strict consensus is shownin ¢ gure3. The maximum- lumpingof taxathat showmorphological similarities. likelihoodanalysis yielded the tree seen in¢ gure4. The Qari et al.(1993)used sequence datato show that the K^Htest signi¢cantly rejected the hypothesisthat the humanmalaria parasite P. vivax consists oftwo forms each erythrocyticforms andforms infectingwhite blood cells withunique circumsporozoite genes, although morpho- fromeach island are each other’ sclosest relativesin logicallythe twoforms arevery similar .One ofthese favourof the monophyleticgrouping of P.azurophilum in forms, called P. vivax-like,is most likely Plasmodium erythrocytes and P.azurophilum inleucocytes (parsimony: simiovale,aparasiteof N ewW orldmonkeys. Escalante et 56steps versus 120steps, p 5 0.0003;maximum like- al.(1995),usingthe same gene,found that twoother lihood: 71044 versus 71260, p 5 0.0001). humanmalaria parasites aregenetically indistinguishable fromthose ofnon-human primates, andsuggested that either host-switchinghas occurred within the last 15000 4.DISCUSSION yearsor even that monkeysmay be serving as reservoir Forthe past 120years, parasitologists have described hosts forthe humanparasites incurrent times. Thus,Qari taxaof malaria parasites usingonly the similarityspecies et al.(1993)and Escalante et al.(1995)have used concept(indeed, this is the standardmethod for taxono- moleculardata to extend the usefulness ofthe similarity mists workingwith any group of protozoan parasites) . species conceptin de¢ ning species of Plasmodium; based Morphologicaltraits such asthe number ofmerozoites ondegree of genetic similarity ordistinctiveness, they per schizont(daughter cells producedby each asexually havechallenged the current taxonomiclabels on these dividingcell) ,shapeof the gametocytes,presence or malariaparasites. Here Ihaveshown that P.azurophilum , absenceof pigment, kind of host cell infected, species of amalariaparasite of Caribbean island lizards, is actually host infected andsymptomatology of infected hosts, have twospecies. Forthe ¢rst time, allthree basictypes of beenused bothto describe species andas characteristics species concepts canbe applied and compared in the inhigher systematic treatments (Garnham1966) .These identi¢cation of acryptic species of Plasmodium. traits arelikely to be unreliable for several reasons. Morphologicalcharacteristics determined from¢ xed, (a) Similarity species concept stainedblood smears maybe altered depending on Underthe lightmicroscope, P.azurophilum in erythro- researchers’ techniques andresult insigni¢ cant di¡ er- cytes andleucocytes are very similar inmorphology . ences inthe appearanceof the parasites underthe light Measurements revealedthat the shapeof the cells does

Proc. R.Soc.Lond. B (2000) Species conceptsand malariaparasites S.L. Perkins2349 notdi¡ er signi¢cantly ,but there areslight di¡ erences in allislands form one clade, as dothose parasites foundin size. Thisis imperceptible tothe eye,however .Theonly leucocytes.The greatest pairwisedi¡ erence observedin phenotypictrait that clearlyseparates them isthe typeof this dataset wasbetween the twoforms onDominica,an host bloodcell infected. Sequencedata for the cytochrome islandin the middleof the archipelago.Thus, there could b genereveal that the twoforms of P.azurophilum di¡er by nothave been multiple evolutionaryorigins on each asmuchas 3.1%.This is comparableto other, recognized island of P.azurophilum inwhite blood cells fromthe ances- species of Plasmodium.Forexample, the same regionof tral linein erythrocytes. this genefrom two species ofbird malaria, P.gallinaceum Over 60years ago, Manwell ( 1936)was prescient in (Asiaticorigin) and Plasmodium elongatum (North stating,`Itseems tome that weshould conceive of a species America)di¡ er by3.4% (Escalante et al. 1998). ofmalaria in genetic terms, aswe do most otherliving things.’Now,armedwith the toolsof moleculargenetics, it (b) Biological species concept seems that weare in a positionto do so. By continuing to Thetwo forms of P.azurophilum arealmost certainly samplepreviously described species ofmalaria parasites reproductivelyisolated. Sampling on multiple islands throughouttheir rangesand collecting DNA sequence revealedthat haplotypesare never shared by the parasites datafrom them, nodoubt some Plasmodium species willbe inred andwhite blood cells; the probabilityof this being foundto actually comprise cryptic species complexes.This aspuriouschance result is vanishinglysmall. Phylogenetic informationwill be invaluable,both for understanding the analysis(S. L. Perkinsand J .J.Schall,unpublished data) populationgenetic structure of Plasmodium asit relates to revealsthat P.azurophilum inwhite blood cells occurs such thingsas drug resistance andvirulence and for withina cladeof typical Plasmodium infectingerythro- unravellingthe broadevolutionary history of this very cytes, soexploitation of white blood cells must bethe diverse groupof parasites. derivedcondition. How did this occur,andwhat prevents the twoforms fromhybridizing ?Thetwo forms often Ithankthe following people for help in the¢ eldand the labora- occurin the same populationof lizards and sometimes tory:Cathy Bliss, Sarah Osgood, Anja Pearson, J os.J. Schall, withinthe same individualhost, o¡ ering an opportunity BrettSchneider, T omSmith, Jason W olfand especially Andrew Wargofor his help with parasitemeasurements. Bill Kilpatrick, forhybridization to occur in the mid-gut ofthe insect SarahOsgood, Jos. J .Schalland Mark Siddall all gave helpful vector.Thetiming of ex£ agellation could di¡ er forpara- commentson the manuscript. The researchwas funded by sites inerythrocytes versus whiteblood cells (dueto the grantsfrom the US National Science F oundation( NSF),the relativeease ofemergence fromeach class ofcell basedon NationalGeographic Society and the NSF -VermontEPSCoR membrane strength).Matingbetween male and female programto J. J. Schall, and a GraduateT rainingGrant from gametes inthe vector’smid-gut occurs veryquickly in NSFto S.L.P . Plasmodium,withinjust afewminutes after ingestion (Carter &Nijhout1977) .Thus,once a genotypeof REFERENCES erythrocyte-infecting P.azurophilum switchedinto white bloodcells it couldhave been reproductively isolated Ayala,S. C. 1970Lizard malaria in California:description of a fromits ancestorform simply by the timingof mating strain of Plasmodiummexicanum andbiogeography of lizard malariain westernNorth America .J.Parasitol . 56, 417^425. events inthe vector. Ayala,S. C. &Hertz,P .E.1981Malaria infection in Anolis Hybridizationbetween species ofmalaria parasite, lizardson Martinique, Lesser Antilles. Rev.Inst. M ed.T rop.Sa ¬ o naturalor experimentallyinduced, has not been reported, Paulo 23, 12^17. althoughthere arenumerous examples of more thanone Baker,A. J.,Daugherty,C. H.,Colbourne, R. &McLennan, species of Plasmodium infectinga givenhost; forexample, J.L. 1 995Flightless brown kiwis of New Zealand possess twoto four species of Plasmodium oftenco- occurin extremelysubdivided population structure and cryptic species humans(Richie 1988;Purnomo et al.1999),asdo lizard likesmall mammals. Proc.N atlAcad. Sci. USA 92,8254^8258. malariaparasites inseveral populations (Schall & Beale,G. H. &Walliker,D. 1 988Biological characterization of Bromwich1 994;Staats & Schall1 996 b).Walliker( 1983) malariaparasites. In Malaria:p rinciplesand p racticeof malar- foundtwo or more species ofrodent malaria commonly iology (ed.W .H.Wernsdorfer& I.McGregor) ,pp.395^409. Edinburgh,UK: Churchill Livingstone. infectingthe same animalin the CentralAfrican Bruna,E. M., Fisher,R. N.& Case,T .J.1996 Morphological Republic,yet based on enzymatic di¡ erentiation he saw no andgenetic evolution appear decoupled in Paci¢c skinks indicationthat the species werehybridizing. Thus,whatever (Squamata:Scinidae: Emoia) . Proc.R. Soc.Lond. B 263, 681^ the mechanism keepingthe twoforms of P.azurophilum 688. separate,the ¢ndingthat twosimilar species of Plasmodium Buckling,A. G.,T aylor,L. H.,Carlton, J .M. &Read,A. F. cancoexist in the same host population,and be repro- 1997Adaptive changes in Plasmodium transmissionstrategies ductivelyisolated, is notextraordinary . followingchloroquine chemotherapy . Proc.R. Soc.Lond. B 264, 553^559. (c) Phylogenetic species concept Carter,R. &Nijhout,M. M. 1977Control of gameteformation Themolecular phylogeny presented here indicatesthat (ex£agellation) in malariaparasites. Science 195, 407^409. P.azurophilum is composedof two evolutionary lineages Cracraft,J. 1983 Species concepts and speciation analysis. Curr. Ornithol. 1, 159^187. andthese lineagescolonized the islandsof the Lesser Creasey,A.M., Ranford-Cartwright,L. C., Moore,D. J ., Antilles independently.If asinglespecies ofparasitecolon- Williamson,D. H., Wilson, R. J.M., Walliker,D. & Carter,R. izedthe islands,then later divergedinto the twoforms, 1993U niparentalinheritance of the mitochondrial gene cyto- then oneach island P.azurophilum inerythrocytes and chrome b in Plasmodiumfalci parum . Curr. Genet. 23, 360^364. leucocyteswould be each other’ sclosest relatives.This is Darwin,C. 1958 Theautobiogra phyof Charles Darwin and selected notthe case.In fact, the parasites inerythrocytes from letters (ed.F .Darwin).NewY ork:Dover Publications.

Proc. R.Soc.Lond. B (2000) 2350S. L. Perkins Species conceptsand malariaparasites

DeMeeu ªs,T .Michalakis,Y .&Renaud,F .1998Santa Rosa Mayr,E. 1963 Animalsp eciesand evolution .OxfordUniversity revisited:or why are there so many kinds of parasites in the Press. `Gardenof EarthlyDelights’? Parasitol.Today 14, 10^13. Paul,R. E.L.,Packer, M. J.,Walmsley,M., Lagog,M., Dye,C. 1991Population genetics of nonclonal, nonrandomly Ranford-Cartwright,L. C., Paru,R. & Day,K.P.1995 matingmalaria parasites. Parasitol.Today 7, 236^240. Matingpatterns in malariaparasite populations of Papua Eisen,R. J.& Schall,J. J .2000Life history of amalariapara- New Guinea. Science 269,1709^1711. site (Plasmodiummexicanum ):independent traits and basis for Posada,D. & Crandall,K. A.1998Model test: testing the variation. Proc.R. Soc.Lond. B 267, 793^799. modelof DNAsubstitution. Bioinformatics 14, 817^818. Escalante,A. A., Barrio,E. &Ayala,F .J.1995 Evolutionary Purnomo,A. S., Solihin,A., Gomez-Saladin,E. & Bangs,M. J. originof human and primate malarias :evidencefrom the 1999Rare quadruple malaria infection in IrianJaya circumsporozoiteprotein gene. Mol.Biol. Evol . 12, 616^626. Indonesia. J.Parasitol . 85, 574^579. Escalante,A. A., Freeland,D. E., Collins,W .E.&Lal,A. A. Qari,S. H.,Shi, Y.P.,Goldman,I. F .,Udhayakumar,V ., 1998The evolutionof primate malaria parasites based on the Alpers,M. P.,Collins,W .E.&Lal,A. A.1993Identi¢ cation geneencoding cytochrome b fromthe linear mitochondrial of Plasmodiumvivax -likehuman malaria parasite. Lancet 341, genome. Proc.N atlAcad. Sci. USA 95,8124^8129. 780^783. Garnham,P .C. C.1966 Malariap arasitesand other haemos poridia . Richie,T .L.1988 Interactions between malaria parasites Oxford,UK: Blackwell. infectingthe same vertebrate host. Parasitology 96, 607^639. Hung,G. C., Chilton,N. B., Beveridge,I., Zhu, X. Q., Schall,J. J .1992Parasite-mediated competition in Anolis lizards. Lichtenfels,J. R. &Gasser,R. B. 1999Molecular evidence for Oecologia 92, 58^64. crypticspecies within Cylicostephanusminutus (Nematoda: Schall,J .J.&Bromwich,C. R.1994Interspeci¢ c interactions Strongylidae). Int.J .Parasitol . 29, 285^291. tested:two species of malaria parasite in aWestAfrican Jordan,H. B. 1970The occurrenceand development of lizard. Oecologia 97, 326^332. Plasmodiummexicanum in thewestern fence lizard Sceloporus Staats,C. M. &Schall,J .J.1 996 a Malarialparasites occidentalis.J .Protozool. 17, 86^89. (Plasmodium) of Anolis lizards:biogeography in theLesser Jordan,H. B. 1975The e¡ect of host constitution on thedevelop- Antilles. Biotropica 28, 388^393. ment of P.£oridense . J.Protozool. 22, 241^244. Staats,C. M. &Schall,J .J.1996 b Distributionand abundance Killick-Kendrick,R. 1978T axonomy,zoogeographyand evolu- oftwo malarial parasites of the endemic Anolis lizardof Saba tion. In Rodentmalaria (ed.R. Killick-Kendrick& W.Peters), island,Netherlands Antilles. J.Parasitol . 82, 409^413. pp.1^52.London: Academic Press. Sturmbauer,C., Opadiya,G. B., Niederstatter,H., Riedmann,A. Kishino,H. &Hasegawa,M. 1989Evaluation of themaximum &Dallinger,R. 1999Mitochondrial DNA reveals cryptic likelihoodestimate of the evolutionary tree topologies from oligochaetespecies di¡ ering in cadmiumresistance. Mol. Biol. DNAsequence data, and the branching order of Hominoidea. Evol. 16, 967^974. J. Mol. Evol. 29, 170^179. Swo¡ord, D .L.1999 PAUP*. Phylogeneticanalysis using p arsimony Lounibos,L. P.,Wilkerson,R. C., Conn,J. E., Hribar, L. J., (*andother methods ),v.4.Sunderland, MA: SinauerAssociates. Fritz,G. N. & Dano¡-Burg, J. A. 1998Morphological, Tamura,F .&Nei,M. 1984Estimation of evolutionarydistance molecular,and chromosomal discrimination of cryptic betweennucleotide sequences. Mol. Biol.Evol . 1, 269^285. Anopheles (Nyssorhynchus)(Diptera: Culicidae) from South TelfordJr, S. R.1975Saurian malaria in theCaribbean: America. J.Med.Entomol . 35, 830^838. Plasmodiumazurophilum sp.Nov .,amalarialparasite with Lymbery,A. J.& Thompson,R. C. A.1996Species of schizogonyand gametogony in bothred and white blood cells. Echinococcus :patternand process. Parasitol.Today 12, 486^491. Int.J .Parasitol . 5, 383^394. Manwell, R.D.1936The problemof species, with special Tibayrenc,M. &Ayala,F .J.1991 T owardsa populationgenetics referenceto the malaria parasites. AnnlsT rop.M ed . 30, ofmicroorganisms: the clonal theory of parasitic protozoa. 435^439. Parasitol.Today 7, 228^232. Manwell, R.D.1943Malaria infections by four species of Walliker,D. 1 983The geneticbasis of diversity in malariapara- Plasmodium in theduck and chicken and resulting parasite sites. Adv.Parasitol. 22, 217^259. modi¢cations. Am. J. Hyg. 38, 211^223. Walliker,D. 1 991Malaria parasites: randomly interbreeding or Mayden,R. L. 1997 A hierarchyof species concepts: the denoue- `clonal’populations ? Parasitol.Today 7, 232^235. mentin thesaga of thespecies problem. In Species:the units of White,G. B. 1974 Anophelesgambiae complexand disease biodiversity (ed.M. F.Claridge,H. A.Dawah& M. R.Wilson), transmissionin Africa. Trans.R. Soc.T rop.Med. Hyg . 68, pp.381^424. London: Chapman & Hall. 278^301.

Proc. R.Soc.Lond. B (2000)