AN OVERVTEWOF CHEMOTAXONOMY AND tTS ROLE rN CREATINGA PHYLOGENETICCLASSIFICATION SYSTEM

COLEENA MANNHEIMER

NationalBotanical Research lnstitute Ministryof Agriculture,Water and RuralDevelopment, P/Bag13184,Windhoek.

INTRODUCTION chemotaxonomicinvestigations have been employed at all levelsof thetaxonomic heirarchy, from subvariety to division (Smithin Street, 1978; Stace, 1980). lt isthought that when Chemotaxonomyis the systematicstudy of chemical the groupsin questiondifferentiated, the abilityto form a variationbetween olant taxa. Evidenceof chemicalvariation chemicalsubstance was retainedby virtueof metabolic has essentiallybeen usedfor classificationpurposes ever processesretained by the group or its ancestors. By since 'folktaxonomies', based on certainobvious plant implicationwe seethat if the pathwayof chemicalevolution characteristicswere instinctivelyemployed by mankind were establishedthen this mightoff er insightto the centuriesago. Thesecategories, such as edibility,taste, evolutionaryhistory of the group,as well as to the colour,smell and medicinalvalue were founded, however understandingof the present-dayrelationships within and unknowingly,on chemicalproperties. As longago as the betweengroups (Erdtmann, cited in Davis& Heywood, first centuryafter Christthe aromaticmints had been 1973). recognisedand groupedtogether by Dioscorides(Jones & Luchsinger,l986). Researchhas shown that there is generallyan inverse relationshipbetween the taxonomicdistribution of a plantsgrew from Awarenessof the chemicalcomplexity of compoundor class of compoundsand its biogenetic the desiresof Europeansfor exoticspices and condiments complexitysuch that if it is biosyntheticallysimple and properties. as wellas investigationsinto their medicinal Early widespreadit may be assumedto be primitive,while those knowledgeabout the subjectwas summarisedin herbals, morelimited in theirdistribution and morecomolex to bio- physiologically andconcentrated on informationabout active synthesisemay be assumedmore advanced (Gershenzon secondarymetabolites such as alkaloidsand saponins & Mabry,1983). Howeverthese assumptionsprove (Jones in Street,1978). simplisticwhen phylogenetic inferences are to be madefrom chemicaldata. Davisand Heywood(1973) indicate the Duringthe eighteenth and nineteenth centuries knowledge followinggeneral complications:- in the field increased,and sometaxonomists made use of severalchemical characteristics inattempts to classifyplants 1. Chemicalcompounds and/or biosynthetic pathways andto demonstratetheir phylogeny. However, although the are not alwaysenvironmentally stable, and so their chemicalcharacters they usedwere recognised, they were presencemay be affectedquantitatively or manifestationsof processesor compoundsnot yet qualitativelyby factors such as temperatureor mineral completelyidentified (Jones & Luchsinger,1986) and so their deficiencies. use was basedon inadequateknowledge and evidence. 2. Biosyntheticknowledge is inadequateabout a great Graduallythe numberof recognisednatural plant products manyof the chemicalcharacters used in taxonomy, increased,extending to includeproteins, nucleic acids and so that parallelismor convergencemight confuse the majorpolysaccharide categories. At the same time perceptionof phylogeny.A compoundmight arise researchinto plantmetabolism revealed similarities and viatwoor moreentirely distinct biosynthetic pathways uniformitiesin the chemicalfunctioning of plants,while (convergence),and its presencewould thus not simultaneouslyhighlighting biochemical peculiarities which necessarilyimply a relationshipbetween taxa in which mightbe taxonomicallyor phylogeneticallysignificant. it occurs.(Gershenzon and Mabry (1983) quote the Successfulattemots were made to correlatethis variation caseof napthoquinones,which may arise in higher with knownclassifications, and manyclaims were made as plants vrafour different pathways). Parallelism may to thetaxonomic merit of variouschemical characters (Smith causesimilar end products to arisein relatedgroups, in Street,1978). Howeverit is only in recentdecades that particularlywhen they are derivedfrom ubiquitous reasonablyrapid surveys of plant extractshave become metabolicintermediates. feasible,due to improvedtechniques of chemicalanalysis and the elucidationof the structuresof many organic 3. ln manycases it is not knownwhether metabolic compounds(Radford et al,1974).Technological advances, pathwaysare reversibleor not, and reversalof a particularlyelectrophoresis and chromatography,have pathwayconfuses the issueof whethera character simplifiedand speededup analyses,and also oftenmade is primitiveor derivedin a specifictaxon. analysisof smalleramounts of materialviable. This is particularlyvaluable when rare herbarium material must be 4. Largeand/or complex molecules are not necessarily used. the mostadvanced. Chemical reduction mav occur in plantsdue to metabolicchanges. It is now generallyaccepted that certaincompounds and relatedsubstances may be characteristicof certain 5. Chemicaland morphologicalfeatures may have taxonomicgroups (Davis & Heywood,1973), and certainly evolvedat differentspeeds due to differingselection

AGRICOLA1998/1999 87 'primitive' pressures,and thus one might find chemical - they are chemicallystable, so that analysisof 'advanced' characterstogether with morphologyin a materialcan be doneVears after the materialis taxon. collected. - they are easilyisolated and identifiedeven from 6. Chemicalvariability occurs - for example,over a smallamounts of plantmaterial. Large numbers geographicrange, or vr,achemical mutants within of plantscan easilyand rapidlybe surveyedfor species. Chemistrymay also vary between flavonoidsusing paper, thin-layer, or oneor two- ontogeneticstages of organsanalysed. djmensionalchromatography, and in recentyears manyuseful results have been achieved (Vickery All theseproblems make it difficultto assessthe relative & Vickery,1981). degreeof advancementof differentcompounds or classes - theyoccur variously but ubiquitously in almostall of compoundsin any giventaxon. According to Davisand plants. Heywood(1973) chemosystematics is of far greateruse in - theycan be usedat alltaxonomic levels in most establishingconsanguinity (kinship) than in elucidationof groupsof plants. phylogenyproper, which involves tracing past evolutionary history. Althoughflavonoids are presentin all higherplants (Vickery& Vickery,1981), they are absentfrom Despitedrawbacks to potentialphylogenetic value, there are bacteriaand the majority of algae.The mostprimitive many instancestoday in whichphytochemical data have groupthat exhibitsthem is the Charophyceae contributedto substantialtaxonomic improvements (eg. (stoneworts),a groupof greenalgae considered to documentationof hybridisation),although they are still be advancedfor a numberof reasons. Simple underutilisedto a largeextent (Stace, 1980). In general, flavonoidshave been found in primitiveBryophyta, restrictedor uniqueoccurrence of compoundsis of greater whilefar morecomplex ones have been isolated from taxonomicvalue than widespreadoccurrence (Davis & the mostadvanced angiosperms (eg.Orchidaceae). Heywood,1973). Broadlyspeaking, four maincategories of charactersare usedin chemosvstematics:- Flavonoidshave been usedto revisethe families includedin the groupCentrospermae. The red anthocyaninand betacyaninpigments are mutually DIRECTLYVISIBLE PARTICLES exclusive,and so tooare the yellow betaxanthins and the carotenoids.A surveyof the Centrospermae Theseare particlessuch as starchgrains and raphides, showedthat while ten of the twelvefamilies included whichare often also regarded as morphologicalcharacters. in the groupcontained betacyanins and/or beta- An exampleof theiruse is in the utilisationof starchgrains xanthins,the Caryophyllaceaeand Molluginaceae to classifythe Poaceae(Polhill and Raven,eds, 1981). containedanthocyanins, in commonwith non- Centrospermaeplants. This argued for theirremoval to a separateorder from a chemosystematicviewpoint PRIMARY METABOLITES (Vickery& Vickery,1981). However,this removalis stilldebated, as their exclusion is indirect contradiction Althoughsome primarymetabolites are utilisedchemo- to the anatomicalevidence, Nevertheless,their systematically,on the wholetheir usefulness is limited,as anomalywas highlightedby theirflavonoid pattern. theyare intermediatesin or productsof essentialmetabolic 'fingerprint' pathways,and as suchare of ubiquitousdistribution (eg. Flavonoidscan be usedas a in some sugarsthat participatein the Calvincycle). Occasionally generawhere it has been found that each species theyaccumulate in unusuallyhigh concentrations or occur has a distinctflavonoid pattern. The Eaptisragenus as unusualstorage products, and it is largelyin such (Fabaceae),for example, contains 62 flavonoids,and instancesthat they are usedtaxonomically (Stace, 1980). eachof its 17 specieshas a characteristicflavonoid pattern. However,closely related genera exhibit severalspecies with almost identical patterns (Vickery SECONDARYMETABOLITES & Vickery,1981).

Theseare very numerous and of morerestricted occurrence Oftenthe geographiccentre of originof a plantcan thanprimary metabolites, which makes them more valuable be deducedfrom its chemicalcharacteristics. as a sourceof taxonomicevidence (Stace, 1980), and a Geraniumspeciesr (Geraniaceae) exhibit a. wide large amountof informationon their distributionin the rangeof flavonoids,with primitivecompounds angiospermsis available.However, the data demonstrate predominatingin CentralEurasian species, and that some secondarycompounds are more usefulthan diminishingin speciesto theeast and west (Vickery others,particularly on a broadscale (Gershenzon & Mabry, & Vickery,1981). 1983).The followingare those groups of compoundsthat havethus far provento be mostuseful generally:- Parentageof naturalhybrids may be deducedif flavonoidbiosynthesis is assumedto be additive Flavonoids (Vickery& Vickery,1981). By recombination,hybrids Jones and Luchsinger(1987) indicate that the arisingfrom specieswith differingflavonoids can flavonoidsare one of the most usefultaxonomic synthesisecompounds found in neitherparent. Such markersfor a varietyof reasons:- chemicalrecombination may resultin the hybrid - they demonstratea wide rangeof chemical exhibitingintermediate exomorphic characters (Davis structures,which have a demonstrablegenetic & Heywood,1973). basisfor their variation.

88 AGRICOLA1998/1999 Terpenoids pathways,including the metabolic intermediates and enzymesinvolved, will have to be established. Despitetheir comparativelywide occurrenceand variety,terpenoids have been used less extensively thanflavonoids in taxonomy, possibly due to difficulty SEMANTIDES of analysis. Howeverthey have been used extensivelyin the chemosystematics of somegroups Thesecomprise DNA, RNA and proteins. Because each of in which they occur (eg. mints,umbellifers). theseis so intimatelyconnected with genetic characteristics, Comparisonof terpenoidcontent between plants has many researchersconsider them to be of immense been facilitatedby gas chromatography,and they taxonomicvalue, as they potentially are. However,the time, have been used to clarifyspecific and subspecific equipmentand know-hownecessary for their effective taxaas wellas geographicraces and hybrids(Jones analysisoften limits their usefulness. & Luchsinger,1987). Stace(1980) mentions three main methods used in Alkaloids proteintaxonomy - electrophoresis, amino-acid sequencing and systematicserology A greatdeal of informationon alkaloidoccurrence is available,and theirdistribution has contributedto Electrophoretictechniques enable proteins to be 'fingerprinted' taxonomicstudies in various groups. However, they byestablishing their relative size, charge and are less chemicallystable than flavonoids,as well isoelectricpoint by separatingthem in variable gel mixtures as being.structurallyand biosynthetically complicaied acrossa voltagegradient (Stace, 1980). Proteinprofiles (Jones& Luchsinger,1987). Due to the lackof producedvra electrophoretic separation and subsequent knowledgeas to the relativeadvancement of the staininghave been used in varioussystematic studies differentbiosynthetic pathways leading to thevarious investigatingpolyploid taxa, as well as at interspecific, skeletaltypes, Iittle attempt has been made to intraspecificand populationlevels. Particularcare and comparedifferent alkaloid types phylogenetically expertiseare required in the use and interpretation of protein (Gershenzon& Mabry,1983). Their contribution to profiles(Jones & Luchsinger,1987). such classificationshas thus been restricted.An exceptionamong the alkaloidsare the bensy- Amino-acidsequencing att€mpts to establishthe lisoquinolinealkaloids, which are important taxonomic variationin the precise sequence of aminoacids in a single markersin the angiosperms. Gershenzon and Mabry homologousprotein throughout a rangeof organisms.This (1983) attributethis to theirbiosynthetic uniformity analysisrelies on the factthat a particularprotein may vary and'coherentdistribution'. to a certainextent without altering its essential function. One moleculeused extensively for this purpose is cytochrome c, Glucosinolates(Mustard oil glucosides) in which79 outof the approximately113 amino acids vary interspecifically,but alterationof even a singleone of the These,together with the alkaloids,have been used other34 destroysthe functioning of the molecule.Generally to dividethe fourfamilies comprising the old order the numberof differencesparallels the relationaldistance Rhoeadalesinto two new orders: betweenthe organismsin traditionalclassifications, but Capparales,containing the Capparaceaeand anomaliesdo presentthemselves, inferring that a measure Cruciferae,which produce glucosinolates. of proteinstructure is not an infallibleguide to degreeof kinshio. Papaverales,containing the Papaveraceaeand Fumariaceae,which produce alkaloids. Certainassumptions are madewhen analysing the results of thistechnioue:- Researchhas shown that glucosinolate patterns may also be used to documenthybridisation as well as It is assumedthat: provideinfrageneric characters (Jones & Luchsinger, - themolecule has evolved yra the minimum number of 1987). mutations - no convergentevolution or back-mutationhas occurred lridoids - differentpositions on the moleculeare equally susceptibleto substitution. Thisgroup of secondarymetabolites is of increasing taxonomicimportance (Gershenzon & Mabry,1983; Theseassumptions weaken the evidencewhen the Jones& Luchsinger,1987), and they show promise sequencingof a numberof homologousproteins yields in the clarificationof relationshipsat variouslevels. conflictingevidence (Stace, 1980). Some researchers have Theyhave also contributed to solvingthe debate over pointedout that to apply cytochromepatterns in chemo- the ancestralprogenitor of theAsteraceae, which do systematicsrequires that one shouldtake intoaccount the not produceiridoids. Several putative progenitors' quantitativeand qualitativeeffects of growthconditions on caseswere weakenedwhen they were shownto thecytochrome content (Kandler & Schleiferin EllenbergeI produceiridoids, leaving only the Campanulaceae, a/, 1980).Stace (1980) suggests that in orderto prevent Araliaceaeand Umbelliferaceae,which do not interpretativemistakes, results from a widerange of proteins, produceiridoids, in the running (Jones & Luchsinger, preferablystudied by a numberof differenttechniques, 1eB7). shouldbe pooledrather than placingtotal relianceon the screeningof a singleprotein. In order to use secondarymetabolites more extensivelyin a systematicallymeaningful way a great Syntheticserology is an immunologicaltechnique relying deal more knowledgeabout their biosynthetic on the relativespecificity of the immunereaction, and the

AGRICOLA1998/1999 RO factthat the degreeof cross-reactivityis proportionalto the 4. Detectionof confirmationof hybridisation. degreeof relationshipbetween the organisms. 5. Providingadditional on/off characters for numerical In plantserology, antisera to antigensfrom various taxa are taxonomy by their presence or absence in taxa. raisedin animals,using various plant extracts (Jones & Luchsinger,1987), and then the antiseracan be usedas a However,as with all other taxonomiccharacters, chemical standardtest againstother plant extracts. The degreeof variationmust constantlybe subjectto criticalappraisal of coagulationthat the other extracts cause in them is usedas techniquesand interpretations.Two major problemsthat a measureof theirsimilarity to the originalantigen. appear to need addressingare the lack of standardisation Refinementsin thetechnique have made this method more of methodologyand the inadequatesampling of groups. speciflcthan it was previously,and serologyhas been extensivelyused throughout the taxonomic levels from above Chemotaxonomyhas undoubtedlymade a big contribution familyto belowspecies, yielding many valuable data (Stace, to taxonomicwork in the past and will most certainlycontinue 1980). to do so in future. However,given the lackof fossilevidence and the need for live materialin some analyses it seems Nucleicacids have not yet beenused very extensivelyin that its contributionto a phylogeneticclassification must plantsystematics due to the complexityof theiranalysis. perforceremain limited. The valuableinformation it offers Mosttechniques are of relativelyrecent origin, and so the is best used in conjunctionwith other sourcesof taxonomic dataaccumulated thus Iar are limited (Jones & Luchsinger, evidenceand thus a multidisciplinaryapproach is required 1987).Theoretically these characters should be ableto solve in order to establisha system of classification which reflects manyphylogenetic problems, firstly because each organism naturalrelationships as accuratelyas possible. has DNA with a uniquebase sequence, and secondly becausethe theoryof evolutionis basedon the premise thatrelated organisms should show similarities intheir DNA REFERENCES whichare not shown by unrelatedspecies (Vickery & Vickery, .1973. 1981 ). DAVIS,PH. AND HEYWOOD, V.H.. PrinciplesofAngiosperm Taxonomy.Robert Kreigler Publ. Co. Inc.,N.Y. The mostuseful technique in thisregard at presentis DNA hybridisation,in whichDNA double helices are inducedto GERSHENZON,J.AND MABRY T.J., 1983. Secondary metabolites unwind.and then allowed to recombinewith each other as andthe higherclassif ication of Angiosperms.Nord. J. Bot. 3: 5-34. wellas similarlytreated DNA f rom other species. This results in some hybriddouble helices being formed, the number JONES,B.J.JNR AND LUCHSINGER,A E., 1987.Plant andf idelity of recombinationstheoretically depending on the Systematics.Mc-Graw Hill Book Co. NewYork. compatibilityofthe two DNA base sequences. Some useful resultshave been obtained which show the potential value KANDLER,O. AND SCHLEIFER, K.H. Systematics of Bacteria.lN: of thismethod, but techniques have not yet been perfected. ELLENBERG.H., ESSER. K.. KUBITZKI, K,, SCHNEPF, Jonesand Luchsrnger (1987) point out that variable results E. AND ZIEGLER,H. (eds),1980. Progress in Botany. havebeen obtained depending on experimentalconditions. Springer-Verlag, Berlin. Some evidencehas suggestedthat in vifroreplication of the DNAtemplate is affected by factorssuch as temperature, POLHILL,R.M. AND RAVEN, PH. (eds), 1981 .Advances in Legume Systematics,Part 2. RoyalBotanic Gardens, Kew. andthe absence of regulatoryphenomena or specific factors that are presentin vivo (Wackernagelin Ellenberget a/, FIADFORD,A E., DICKISON, W.C.. IVASSEY, J.R. AND BELL, C,R,, .1974. 1980),and it seemswell possible that this might just as well VascularPlant Systematics. Harper and Row.N.Y. appiyto recombinationalso. SlVlTH,P.M. Chemical Evidence in PlantTaxonomy. lN. Street, H.E., Othertechniques have been used to investigateDNA and 1978. Essays in PlantTaxonomy. Academic Press, London. RNA,but results,according to Stace(1980), are of limited application.He suggests that advances in gene cloning and STACE,C.A., 1980. Plant Taxonomy and Biosystematics.Edward geneticengineering may lead to more extensiveuse of Arnold(Publishers) Ltd., London. nucleicacid characters in taxonomy, but a potentialdrawback VICKERY,lV.L. AND VICKERY,8.,1981. Secondary Plant to theirextensive use in phylogenyis that livematerial is Metabolism.The MacMillanPress Ltd.. London and oftena orereouisite. Basingstoke.

Chemicalvariation is of considerabletaxonomic value in WACKERNAGEL,W. REPLICATION.IN: ELLENBERG,H., severarways:- ESSER,K., KUBITZKI, K., SCHNEPF, E, AND ZIEGLER. H. (eds),1980. Progress in Botany.Springer-Verlag, Berlin. 1. Confirmationor supportof putativeclassifications derivedfrom other sources of taxonomiccharacters. suchas morphology. a. Resolutionof problemswhere relationships based on otherevidence are ambiouous or conflictino.

Providingevidence to suggestmore natural positioningof anomaloustaxa, as well as to separate taxa. Often the presenceof anomaloustaxa in a groupis accentuatedby theirchemical peculiarities.

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