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New Z ealand Journal of Botany, 1993, Vol. 31 : 43-58O028-825X/93/3101-O043 $2.50/0 The Royal Society of New Zealand 1993 43

Systematics of New Zealand Inuleae (Compositae-Asteraceae)3Num erical phenetic analysis of leaf anatomy and flavonoids

ILSE BREITWIESER*JOSEPHINE M. WA RD

Department of Plant and Microbial SciencesUniversity of CanterburyPrivate Bag 4 800Christchurch, New Zealand*Present address: Botanischer Garten und BotanischesMuseum Berlin-Dahlem, Konigen-Luise-Strasse 6-8, D-1000 Berlin 33 , Federal Republic of Germany.Abst r a c t Forty-f ive species of Inuleae(Compositae), predominantly from New Zealandand Tasman ia, were subjected to num erical pheneticanalysis using 49 leaf anatomy and 38 leaf flavonoidcharacters. Results indicate that on the basis ofoverall similarity in this suite of characters: (1)Leucogenes is a good genus; (2) an undescribedtaxon of Inuleae is a distinct monotypic genus; (3)New Zealand species currently placed in Anaphalis,Cassinia, and Ewartia are generically distinct butare referred to the wrong genera; (4) similaritybetween species of Ewartia an d Gnaphaliumindicates that the boundary between these generaneeds to be reassessed; (5) both Helichrysum andRaoulia are heterogeneous and need revision; (6) inNew Zealand, Pseudognaphalium is distinct fromother gnaphalioid genera.Keywords Compositae; Asteraceae; Inuleae;Gnapha l i inae ; Anaphalis; Cassinia; Ewartia;Gnaphalium; Haastia; Helichrysum; Leucogenes;Pseudognaphalium; Pterygopappus; Raoulia; leafanatomy; f lavonoids; taxonomy; systemat ics;numerical p henetics; New Zealand; Tasm ania

INTRODUCTIONThis study uses analysis of chemical and an atomicalleaf data to elucidate taxonom ic relationships in theNew Zealand Inuleae*. A representative selectionof species of all indigenous genera except Craspediais analysed as well as critical species from Tasm aniaand a single species of Anaphalis from theHimalayas, included for comparison with the NewZealand species recently transferred by Webb (1987)to this genus. Taxa examined are listed in Table 1.Of the nine genera of the Inuleae which areindigenous to New Zealand, Craspedia is part of theAngianthus complex centred in Australia and refer-able to Bentham's (1873a) subtribe Angianthinae.The remaining eight genera all fall within Ben tham 'ssubtribe Gnaphaliinae, and are in the groups ofgenera clustered around Anaphalis, Gnaphalium,and Helichrysum (Merxmiiller et al. 1977)^.The classification of New Zealand species ofGnaphal i inae has been s t rongly affec ted byEuropean ideas, especially those of Bentham andHooker. In the nineteenth century, most herbaceousNew Z ealand species were referred to one of the twola rge , widespread gene ra Gnaphalium andHelichrysum, distinguished by the ratio of female

*Anderberg (1989) regards the tribe Inuleae sensu lato asparaphyletic. He has divided it into three tribes, with allNew Zealand genera falling into the largest of these, theGnaphalieae. How ever, paraphyly of the Inuleae is not yetsufficiently established to warrant abandoning the traditionalconcept of this tribe (Bremer et al. 1992). We do not acceptthe revised subtribes proposed for the Gnaphalieae byAnderberg (1991), which separate closely related(hybridising as well as phenetically similar; Ward 1981,1993b) New Zealand genera at the subtribal level. Genericname changes proposed by Anderberg (1991) are notedbut not used in this paper. They will be discussed andrecommendations will be made regarding their use in aforthcoming revision of New Zealand Inuleae.

B92012Received 31 March 1992; accepted 3 February 1993

subtribal nam e Gnaphaliinae is used throughout thispaper in the sense of Bentham, no t the much broader senseof Merxmiiller et al. (1977).


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44 New Zealand Journal of Botany, 1993, Vol. 31Table 1 Species included in this paper.Anaphalis A. triplinervis Himalayas

A. keriensisA. rupestrisA. subrigidaA. trinervis

New ZealandNew ZealandNew ZealandNew ZealandCassinia C. aculeataC. longifolia TasmaniaTasmania

C. fulvidaC. leptophylla

New ZealandNew ZealandEwartia

Helichrysum

subg. Misturasubg. Psychrophyton

E. catipesE. meredithiae

R. cinereaR. species 1R. petriensisR. grandifloraR. hectoriiR. bryoidesR. eximiaR. species 2

TasmaniaTasmania

Gnaphalium sect.Euchiton

E. planchonnE. sinclairiiG. involucratumG. mackayiG. nitidulumG. traversii

TasmaniaNew ZealandNew ZealandNew ZealandNew ZealandNew Zealand

sect.Ozothamnus H. backhouseiH. obcordatumH. lanceolatumH. coralloidesH. depressantH. dimorphumH. intermediumH. parvifolium

TasmaniaTasmaniaNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew Zealand

LeucogenesPseudognaphaliumRaoulia

subg. Laphangiumsubg. Raoulia

sect.Xerochlaena

H. bellidioidesH. fllicauleL. grandicepsL. leontopodiumP. luteoalbumR. hookeriR. tenuicaulisR. glabra

New ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew ZealandNew Zealand

Inuleae genus 1 undescribed species New ZealandHaastiaPterygopappus

H. pulvinarisH. sinclairii

New ZealandNew ZealandP. lawrencei Tasmania

Solid lines show present taxonomic limits. Dotted lines show limits suggested by this study.


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Breitwieser & WardN.Z. Inuleae 45to hermaphrodite florets in the capitulum . The effectsof this have been to group the New Zealand taxainto widely distributed genera with which they havelittle in common and to obscure differences amongthe New Zealand taxa them selves. Classification hasalso been ham pered by the overlapping distributionsof critical character states among N ew Z ealand taxa.A given species may possess diagnostic characterstates of several genera, with its generic positionchanging as different characters are emphasised.

Of the genera of Gnaphaliinae indigenous to NewZealand, Anaphalis is characterised by conspicuouswhite involucral bracts, dorsal sweeping hairs allalong the style arms, and subdioecy. It is a mainlyAsian genus with four species, all endemic, in NewZealand. The N ew Zealand species were classifiedby Bentham (1873b) as Gnaphalium sec t ionAnaphalioides, connecting Gnaphalium toAnaphalis and Helichrysum. Separate generic statushas been proposed or suggested for them(Kirpichnikov & Kuprijanova 1950; Drury 1970;Merxm iiller et al . 1977; Anderberg 1991), but Webb(1987) transferred them to Anaphalis, pending moredetailed investigation of their relationships. Severalauthors (Drury 1970, 1971; Ward 1981 , 1993b;W ebb 1987) have noted the similarity between thesespecies and the endemic Helichrysum bellidioides,which was not formerly classified with them inGnaphalium because it lacked the preponderance offemale florets characteristic of that genus.Cassinia is an Australasian genus of some 21species , charac ter i sed by a shrub habi t andhomogamous capitula which have white involucralbracts and are clustered in secondary inflorescences.I t i s d is t inguished f rom Helichrysum sec t .Ozothamnus by having receptacular paleae amongthe florets. The New Zealand species (five in Allan1961, but reduced to one by Webb 1988) are allendem ic. Sometimes they lack receptacular paleae.It has been suggested (H ooker 1864; Hilliard & Burtt1973; Breitwieser 1990, in press) that they are closerto Aust ra l ian species of Helichrysum sec t .Ozothamnus than to Cassinia.

Ewartia has four Australian species which havea mat habit, terminal capitula which are solitary ora few in a cluster, subdioecy, and dorsal sweepinghairs all along the style arms. The single NewZealand species, which is endemic, and which wastransferred from Helichrysum by Cheeseman (1925),has none of these features and appears to be palpab lymisp laced in Ewartia (Ward 1981 , 1993b) .Anderberg (1991) has proposed generic status forthe New Zealand species, naming the new genus

Ewartiothamnus. The integri ty of Ewartia inAustralia has also been questioned (Ward 1981,1993b; Anderberg 1991).Gnaphalium sensu lato has traditionally been avast, heterogeneo us, widespread genus characterisedby its herbaceous habit, usually tomentose leaves,and capitula with predominantly female florets. TheNew Zealand species in Allan (1961) were shownby Drury (1970) to comprise three distinct groups.The "achyroclinoid g roup", w ith divided stereomes,had one ind igenous spec ie s , G. luteoalbum,subsequently transferred to Pseudognaphalium byHilliard & Burtt (1981). The "anaphalioid group"had four indigenous species, transferred by Webb(1987) to Anaptialis as out l ined above . The"gnaphalioid grou p" remained in Gnaphalium, withall New Zealand species fal l ing within sect .Euchiton, which has i ts greatest species con-centration in this country and for which separategener ic s ta tus has recent ly been proposed(Anderberg 1991). It is characterised by a stolon-iferous habit, achenes with paired papillae (Drury1970), pappus hairs with basal cilia, and capitula interminal and sometimes axillary involucrate clusters,or solitary. Of the 14 indigenous species, two, G.mackayi an d G. nitidulum, differ in their non-stoloniferous mat habit and achenes with imbricaterather than paired papillae. Bentham (1873b) notedthat G. nitidulum is scarcely distinguishable fromEwartia (then Raoulia) planchonii of Tasmania.About half the New Zealand species are endemic,the remainder being shared with Australia and one,G. involucratum, also with Taiwan, Java, and thePhilippines (Webb 1988).

Helichrysum, like Gnaphalium, has been a verylarge widespread genus, to which a heterogeneousassortment of New Zealand species with pre-dominantly hermaphrodite florets has been referred.Of these, two have been transferred to Leucogenes(Beauverd 1910), one to Raoulia (Beauverd 1912),and one to Ewartia (Cheeseman 1925). There remaintwo species in the herbaceous perennial sectionXerochlaena and seven in the mainly woody sectionOzothamnus, which is recognised at generic levelby Anderberg (1991). Of the seven species in sect.Ozothamnus, one, Helichrysum lanceolatum, is ashrub with broad, spreading leaves and capitula infasciculate clusters. It is quite unlike any othergnaphalioid New Zealand taxon. A further five arewhipcord shrubs with small, imbricate, appressedleaves and solitary, sessile capitula. The hab it of theremaining species, Helichrysum dimorphum, isrema rkable. N ot only is the plant scandent (the only


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46 New Zealand Journal of Botany, 1993, Vol. 31example of this habit in the New Zealand Inuleae)but the shoot apex produces alternating and quitedifferent leaf forms, one scale-like, and appressedto the stem, the other broad, and strongly deflexedbasally. H. dimorphum grows with and is inter-mediate in morphology between H. depressum andH.filicaule. It is now established (Allan 1961) thatit is not a direct hybrid of these two species assuggested by Wall (1920), but the possibility ofhybrid origin remains.

Leucogenes was erected by Beauverd (1910) forthe two species (both endemic to New Zealand) ofHelichrysum sect. Leontopodioides, which arecharacterised by capitula like those of Helichrysumsect. Ozothamnus but arranged, like those ofLeontopodium, in a dense terminal cyme surroundedby a ring of conspicuous bracts.Raoulia is a large, endemic New Zealand genusof small-leaved mat or cushion plants with solitary,sessile, terminal capitula (for an account see Ward1981, 1982a). I t has long been regarded asheterogeneous, with the subgenera Raoulia andPsychrophyton sometimes given generic status(Beauverd 1910; Anderberg 1991), but more usuallyregarded as sections (Bentham 1873b; Kirk 1899;Cheeseman 1906) or subgenera (Beauverd 1912;Cheeseman 1925; Allan 1961).Inuleae genus 1 (CANU 35556) is an unpublishedmonotypic genus (Ward unpubl.) described by Allan

(1961, p. 714) under Leucogenes incertae sedis. Hecompared it with Haastia sinclairii and Leucogenesand suggested a possible hybrid origin. It differsfrom both these genera in posses sing, in addition toterminal capitula, lateral capitula in the axils of theupper foliage leaves.The other genera included in this study areHaastia and Pterygopappus. Haastia was originallyplaced with the "gnaphalioid Compositae" (Hooker1864) but included in the tribe Astereae by Kirk(1899), Cheeseman (1906, 1925), and Allan (1961)because it lacks the tailed anthers of the Inuleae.Merxmlil ler et al . (1977), without presentingreasons, transferred the type species (H. pulvinaris)to the Inuleae and commented that the other twospecies "seem to represent quite another genus".Pterygopappus is a monotypic Tasmanian endemicgenus , a small-leaved mat plant with solitary,terminal capitula and florets with a few broad,flattened, serrulate pappus hairs. It was included intheir "Gnaphalium group" by Merxmiiller et al.(1977), who suggested a position close to Haastiapulvinaris, whereas Anderberg (1991) associated itwith Raoulia subg. Psychrophyton (as Psychrophyton).

The present study applies techniques of numericalphenetic analysis to chemical and anatomical leafcharacters in New Zealand Gnaphaliinae. This is oneof a series of studies including taxonomic, bio-systematic, phenetic, and cladistic approaches andleading to a full taxonomic revision of this group.

MATERIAL AND METHODSAll material was collected in the field betweenJanuary and March of 1987-89, except material ofAnaphalis triplinervis which was taken fromcultivation. Details of voucher specimens are givenin Appendix 2. Names follow A llan (1961) or Webb(1987, 1988) for New Zealand species and Curtis(1963) for Tasmanian species. The genus of Inuleaeand the two species of Raoulia for which publishednames do not exist are designated by num bers withintheir ranks and referenced to a CANU herbariumnumber. They are Inuleae genus 1 (CANU 35556),Raoulia species 1 (an alpine mat plant with blackishleaves, CANU 34359), and Raoulia species 2 (asmall cushion plant of Central Otago mountains,CANU 35559)*.

For the numerical analyses, three data matricesof 45 taxa with, respectively, 49 leaf anatomycharacters, 38 flavonoid characters, or all 87characters, were used. (Flavonoid distribution isshown in Table 3. Anatomy characters are listed inAppendix 1.)For leaf anatomy studies, leaves w ere fixed in 3%glutaraldehyde and emb edded in 1B-A (PolysciencesInc. embedding kit) as described in Breitwieser(1990, in press). Transverse sections of 4 iimthickness were stained in aqueous methylene blue/azure blue. Detailed comments on the leaf anatomycharacters and descriptions of the leaf anatomy ofeach taxon examined are given in Breitwieser (1990,in press).For leaf flavonoid analysis, two-dimensionalpaper chromatograms of crude leaf extracts wereprepared following the techniques of Mabry et al.(1970) and Markham (1982). Two-dimensionalpaper chromatograms (2D-PCs) having spots withlow m obility in the solvent system TBA/15% HOAcwere rerun in 50% HO Ac. The identity of the spots

*In Ward (1981,1982a, b) and Breitwieser (1990, in press),Raoulia species 1 and 2 are referred to, respectively, asRaoulia sp. "M" and Raoulia sp. "L". In Breitwieser (1990,in press), Inuleae genus 1 is referred to as genus "Z".


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Breitwieser & WardN.Z. Inuleae 47was determined by their R f values, by their colourunder UV (366 nm) before and after fuming withammonia, by spraying with a 1% solution ofNaturstoffreagenz-A in methanol, and by co-chromatography, as well as, in some specimens, byUV absorption spectroscopy and by hydrolysis.These procedures give results considered sufficientlyreliable for use in preliminary systematic com-parisons. Problems in the use of phenolic spotpatterns have been discussed fully elsewhere (e.g.,

Table 2 R f values and fluorescence characteristics ofleaf flavonoids in 45 species of Inuleae.

Spotno .

Colourunder UV

ColourunderUV+N H 3

Rfvalue(xlOO)inTBA

Rf value(xlOO)in 15%HOAc

Rf value(xlOO)in 50%HOAc

1234567891011121314151617181920212223242526272829303132333435363738

purplepurplepurplepurplepurplepurplepurpleyellowpurplepurplepurplepurplepurplepurplepurplepurplepurplepurpleyellowyellowpurplepurpleyellowyellowpurplepurplepurplepurplepurplepurpleyellowyellowpurpleyellowpurplepurpleyellowpurple

greengreengreengreengreengreengreenyellowyellowpurpleyellowyellowgreengreenpurpleyellowgreenpurpleyellowyellowgreengreenyellowyellowyellowpurplepurpleyellowgreengreenyellowyellowyellowyellowyellowpurpleyellowpurple

4737384533405260687720371612271585909080233727554173925155614159636569683784

4539485961201622220304765513321022>10>10>10>10

>10>1042>10>10>10>10>10>10>1022

25

615423323372742372330435250

Adams 1974; Harborne 1975; Harborne & Turner1984; Breitwieser 1990). For the data matrix, theflavonoid characters were coded for presence andabsence. Where the presence of a spot varied withinone taxon, or the identity of one spot could not bedetermined, the s ta te was entered as "nocomparison".Similarities between the species were calculatedusing Gower's general coefficient of similarity forqualitative and quantitative characters (Gower1971). The similarity values were clustered byUPG MA (the unweighted pair group method usingarithmetic averages; Sokal & Michener 1958), whichgenerally gives the least amount of distortion of asimilarity matrix (Rohlf 1970; Sneath & Sokal 1973;Ward 1981). The program used for the numericalanalyses was "Gower", by C. M. Frampton, G. A.Findlay, and J. M. W ard, C hristchurch.

RESULTSFlavonoidsExamination of the individual 2D-PCs under UVlight revealed the presence of 65 different UV-fluorescent or absorbing sp ots. These were, with theexception of the 14 bright yellow fluorescent spots,probably phenolic or flavonoid in nature (Markham1982). On the 2D-PCs, all spots relevant to thepresent study typically appeared dark purple oryellow. They are distinguished by their colourchanges in NH3 vapour and by their R f values inTBA, 15% aqueous acetic acid, and 50% aqueousacetic acid (Table 2). Other blue fluorescent spots(a-k) were encountered in almost all taxa. They areprobably simpler phenolics such as cinnamic acidsand their glycosides. Their 2D-PC pattern is ofteninconsistent and for this reason no attempt has beenmade in this study to use these components in ataxonomic interpretat ion. The only compoundappearing as a red spot, identified as a cyanidin-O-glucoside, was excluded for the same reason. Figu re1 shows the m aster composite chromatogram of allcompounds derived from all individual chromato-grams, while Fig. 2 shows the master compositechromatog ram of flavonoids w hich were used in theanalyses. The chrom atograms characterised by spotswith low mobility in standard solvent systems werererun in 50% acetic acid to obtain better resolution.Only the spots with low mobility can be seen here,as shown in the master chromatog ram (Fig. 3), sinceall the other spots ran with the solvent front.

The semi-quantitative distribution of the leaf


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Table 3 Semi-quantitative distribution of leaf flavonoids in 45 species of Inuleae. KeyRelati ve concentrat ion (as jud ged visually): +++ = high, ++ = moderate, -p..+ = low, = trace, ? = inconsistent

SpotTaxon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38Anaphalis keriensis + + + + + + , + A. rupestris + + + + + + . + + + .A. subrigida ++ + . ++ .A trinervis + +++ . A. triplinervis + + + . Cassinia aculeata + . . ++ +++ +++ . ++ ++ . .C.fiilvida . . ? + . . . . + + + + + + + + + + + +C. leptophylla . . ? + . . . . + + + +C. longifolia + +++ ++ . + + . .Ewartia catipes + + + . . + + + + . . + . . +E . m e r e d i t h i a e +++ + ++ . . + + . . . E. planchonii + + + + . . + + + . + +E. sinclairii . + . . . +++ Gnaphalium involucratum + + + + . . + . . + . . . .G. mackayi + + . . + . G. nitidulum ++ + . . +G. traversii + + + ?Haastia pulvinaris +++ . +H. sinclairii + + + . ? . . . . + + + +Helichrysum backhousei . . . + +H. bellidioides +++ . +++ +++ ++H. coralloides ? ? . . . ? ++H. depressum +++ +++ +++ ? ^H. dimorphum +++ ^H. filicaule + + + . + + . + + fsjH. intermedium +++ ++ + JH. lanceolatum ? ^H. obcordatum . . . +++ . . . . + + + + +++ +++ J5H. parvifolium + +++ +++ l_^Leucogenes grandiceps . + + + + +++ +++ . ++ ++ . + OL. leontopodium . + ? ? . . . + + + + + + + + . + + + . . . ++ + + + + + + + + . . . gPseudognaphalium luteoalbum . . . . aPterygopappus lawrencei + ++ . . ? +++ + + ++ ^Raoulia bryoides . . + + l~f>R . cinerea . . . WR. eximia + + . + + + + + QR. glabra + . . . . ? +++ +++ . + . + . +++ pR . g r a n d i f l o r a + + + + . . + + + . + + + . + . . + + + . . . . f ~R. hectorii . . + + + ? . . . + + + gR . h o o k e r i . + + + . . + . . + + + . . . + . . . .


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Breitwieser & WardN.Z. Inuleae 49

3UI&1 C M

1 TBA - ^Fig. 1 Master composite chromatogram of all compounds in the leaves of 45 species of Inuleae. Spot colour key:continuous outline - purple or yellow spot; broken outline - blue spot; dotted outline - bright yellow fluorescent spot;hatched spot - red spot. Numbering and lettering: all flavonoids are numbered (see Table 2, 3). Lettering refers to thefollowing: a - k,other phenolic compounds; R, rutin marker.

flavonoids shows that all of the 45 species examinedare distinguished by their flavonoid pattern (Table3), although in some the differences are minor or ofa quantitative nature only. Limited investigation ofinfraspecific flavonoid variation indicated it is eitherabsent or minor and of a quantitative rather than aqualitative nature. Relative intensities of spots referonly to the pattern of spots for the taxon underconsideration and are not comparable between taxa.Three major groups of spots can be distinguished(Fig . 4) . These are the group of f lavonol-3-glycosides in the centre of the 2D -PC (spots I), thespots with little mobility in standard solvent system snear the origin (spots II), and the spots w ith little orno mobility in 15% aq. acetic acid, but very highmobility in TBA (spots III). Spots III may have been

surface flavonoids since they could not be detectedon the chromatograms if the leaves were shaken inchloroform before extraction in methanol. Specieswith only spots I are the New Zealand Anaphalisspp., Helichrysum bellidioid.es an d H. filicaule,Haastia pu lvinaris and H. sinclairii, Rao ulia eximiaand Raoulia species 1, and the TasmanianPterygopappus lawrencei. The New Zealand speciesof Cassinia and the Tasmanian species ofHelichrysum studied have spots III in comm on. Thespecies which are characterised by spots II only areAnap halis triplinervis, Pseudo gnaph aliumluteoalbum, Raoulia bryoides, R. grandiflora,Raoulia species 2, all Tasmanian Ewartia spp., andthe New Zealand Gnaphalium species studied exceptG. traversii. Helichrysum lanceolatum is the only


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50 New Zealand Journal of Botany, 1993, Vol. 31

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Breitwieser & W ard N.Z. Inuleae 51

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7===numerical phenetic analysis of leaf anatomy and flavonoids=tlc

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