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P1. Syst. Evol. 208:121-138 (1997)

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Phylogenetic relationships of Pseudopanax species (Araliaceae) inferred from parsimony analysis of rDNA sequence data and morphology A. D. MITCHELL and S. J. WAGSTAFF Received May 1, 1996 Key words: Araliaceae, Cheirodendron, Meryta, Pseudopanax. - Internal transcribed spacer

(ITS), morphology, parsimony analysis, phylogenetic relationship, rDNA sequence data. Abstract: Sequence data from internal transcribed spacer (ITS) regions of rDNA and data

from morphology, cytology and wood anatomy are used to study phylogenetic relationships in Pseudopanax. The molecular and non-molecular data are analysed as independent data sets and in combination using parsimony. Results supported the conclusion that the genus Pseudopanax is potyphyletic. Pseudopanax species emerge in two major monophyletic groups. The Anomalus group contains Pseudopanax anomalus, P. edgerleyi, and P. simplex; these species share a common ancestor with Cheirodendron trigynum and more distantly with Pseudopanax gunnii. The second major group consists of two smaller groups: the Arboreus group, including Pseudopanax arboreus, P. colensoi, P. kermadecensis, P. laetus, and P. macintyrei, and the Crassifolius/Discolor group, including P. chathamicus, P. crassifolius, P. discolor, P. ferox, P. 9illiesii, P. lessonii, and P. linearis. Meryta species are close relatives of the Pseudopanax Arboreus and Crassifolius/Discolor groups.

The Araliaceae comprise approximately 50-55 genera and approximately 1525 species (D. G. FRODIN, pers. comm.). A family of mainly woody trees or shrubs, with fewer lianes and perennial herbs, the Araliaceae are thought to be paraphyletic by exclusion of the herbaceous Apiaceae (JUDD & al. 1994, PLUNKETT & al. 1996). Monophyly of the Araliaceae/Apiaceae is supported by compound leaves with sheathing base, stipules, umbellate inflorescences, minute calyx, inferior ovary capped by nectariferous tissue, and fleshy fruits with well developed endocarp (JUDD & al. 1994). While the Apiaceae are largely concentrated in temperate areas, Araliaceae are widely distributed in tropical and subtropical regions, being found mostly in southeastern Asia and land masses of Gondwana origin (I:~-IIUPSON1979). Few araliads have extended their range to include cool-temperate regions (PHILIPSON 1979). The diversity of Araliaceae found in temperate New Zealand is remarkable, with Pseudopanax, Schefflera, Meryta, and Stilbocarpa represented. Pseudopanax includes 21 species, 15 in New Zealand and the others in New Caledonia, Tasmania,

122

A.D. MITCHELL& S. J. WAGSTAFF:Phylogeny of Pseudopanax

China, and Chile (PHILIPSON 1965). The taxonomy of Pseudopanax has been subject to considerable debate, species having been placed in Panax by HOOKER (1853: 94), Pseudopanax by KOCH (1859), Raukaua and Nothopanax by SEEMANN(1866, 1868), and Neopanax by ALLAN (1961). Groups of Pseudopanax species have been recognised on the basis of leaf morphology (CHEESENAN 1906, 1925), floral morphology, wood anatomy, numbers of locules in the ovary, heteroblastic form (SOWR 1957), shoot morphology (PmL~PSON 1971), and wood anatomy, especially of the vessel elements (BUTTERFIELD& al. 1984). Pseudopanax has not previously been studied using phylogenetic methods, and there is presently little evidence to support the m o n o p h y l y of the genus (W. R. PHILIPSON,pers. comm.). At the commencement of this study the closest relatives of Pseudopanax were not known, although HARMS (1894-1897) placed Nothopanax ( = Pseudopanax) with Cheirodendron, Acanthopanax ( = Eleutherococcus), and Astrotricha on the basis of their shared possession of palmate, palmately lobed, or simple leaves, articulating pedicels below the flower, 2 - 4 locules per ovary, and smooth endosperm. This study poses three questions: (1) Does Pseudopanax represent a monophyletic group? (2) What are the close relatives of Pseudopanax? and (3) Can monophyletic groups be determined within Pseudopanax?

Materials and methods The study group. The 33 taxa included in this study are listed in Table 1 with details of their provenance, vouchers and/or living plant numbers. Leaf and/or fruit material was usually either extracted on the day of collection or stored in silica gel before extraction, although herbarium specimens were also used as a source of DNA for analysis. The study group included all New Zealand Araliaceae, i.e. 15 species of Pseudopanax, plus Schefflera digitata, Meryta sinclairii, and three species of Stilbocarpa. Pseudopanax species included from outside New Zealand were P. davidii from China and P. 9unnii from Tasmania. Species not considered were the South American P. laetevirens(GAY)HARMS,the Chinese P. delavayi (FRANCH.)PHILIVSONand P. rosthornii(HARMS)PHILIPSON,and the New Caledonian P. scopoliae (BAmL.) PmLIVSON. Additional representatives of Pacific AraIiaceae included ArthrophyIlum angustatum from New Caledonia, Meryta pauciflora from the Cook Islands, and Polysciasfruticosa with unknown centre of endemism, but according to LOWRY(1989) present in Vanuatu. Pittosporum dallii was included in the study to provide a root for the phylogenetic tree, i.e. this taxon was designated as the only outgroup for phylogenetic analysis. A relationship between the Pittosporaceae and the AraIiaceae/Apiaceae was inferred by XIANG& al. (1993) and PLUNrd~TT& al. (1996) on the basis of rbcL sequence data and by JUDD & al. (1994) and the authors contained therein using morphology. Sequencing protocol. Total DNA was extracted using a modified CTAB method of DOYLE8,=DOYLE(1987). DNA was amplified in two steps using the polymerase chain reaction (PCR). Initially i gl of the total DNA extract was used as a template for the amplification of double-stranded DNA (dsDNA). Single-stranded DNA (ssDNA) was then generated by asymmetric PCR amplification using 10 gl of the amplified dsDNA as a template and only one of the two primers (KALTENBOCK& al. 1992). Excess primers and salts were removed from the ssDNA by precipitation with isopropanol in the presence of 2.5 M NH4Ac followed by a 70% EtOH wash. Both the forward and reverse strands of the internal transcribed spacer (ITS) region of nuclear encoded ribosomal DNA were sequenced using the protocol of BALDWIN (1992).

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5 locules (BENTHAM & HOOKER1867, HARMS1894-1897, CHEESEMAN1925, L11942, ALLAN1961, PmLIPSON1965) 11. Nature of style arms: free, connate at base/free at tips, completely fused (BENTHAM & HOOKER1867, HARMS1894--1897, CHEESEMAN1925, L~ 1942, ALLAN1961, PHILIPSON1965) Androecium 12. Anther attachment: dorsifixed, basifixed (BENTHAM& HOOKER1867, HARMS1894-1897, CHEESEMAN1925, ALLAN1961, PHILIPSON1965) Inflorescence 13. Petal or stamen number per flower: 5, > 5, 4 (BENTHAM• HOOKER 1867, HARMS 1894-1897, CHEESEMAN1925, LI 1942, ALLAN1961, PHILIPSON1965) 14. Petal type in bud: valvate, imbricate (BENTHAM& HOOKER 1867, HARMS1894--1897, L~ 1942, PHIHPSON 1965) 15. Gyn°ecial d°rsal vascular bundles: united with the peripheral bundles' separate fr°m the peripherals (EYDE & TSENG 1971) 16. Gynoecial ventral vascular bundles: homocarpellous, heterocarpellous, solitary central strand (EYDE& TSENG 1971) 17. Inflorescence as a whole: simple umbel (2 orders of branching), compound raceme (3 orders of branching), compound umbel (3 orders of branching), panicle (4 orders of branching), cyme, corymb (BENTHAM& HOOKER1867, HARMS1894-1897, CHEESEMAN1925, LI 1942, HUTCHINSON1967, ALLAN1961, FROD~N1982, PHILIPSON1965) 18. Arrangement of flowers: umbellulate, capitulate, racemulate, spiculate (BENTHAM & HOOKER 1867, HARMS 1894--1897, LI 1942, HUTCHINSON1967, FRODIN 1982, PHILIPSON 1965)

Phylogeny of Pseudopanax

127

19. Relation to associated vegetative parts: terminal, axillary (BENTHAM& HOOKER 1867, HARMS1894--1897, CHEESEMAN1925, L11942, HUTCHINSON1967, ALLAN1961, FROBIN1982) 20. Flower sessile or pedicellate: sessile, pedicellate (BENTHAM& HOOKER 1867, HARMS 1894-1897, CHEESEMAN1925, LI 1942, HUTCHINSON1967, ALLAN1961) 21. Pedicel type: articulating below flower, continuous with flower, not applicable (BENTHAM & HOOKER 1867, HARMS 1894--1897, LI 1942, HU 1980) 22. Bracts: present, reduced or absent (BENTHAM& HOOKER 1867, HARMS 1894 1897, LI 1942, HUTCHINSON1967) 23. Sexuality: hermaphrodite, polygamous, monoecious, dioecious (BENTHAM& HOOKER 1867, HARMS 1894 1897, CHEESEMAN1925, HUTCHINSON 1967, ALLAN 1961, EAGLE 1975, SALMON1980, LOWRY1990) 24. Endosperm surface: smooth, ruminate rugose (BENTHAM& HOOKER 1867, HARMS 1894-1897, CHEESEMAN1925, HUTCHINSON 1967, ALLAN 1961, FRODIN 1982, PHILIPSON 1965) 25. Lateral face of endocarp: flat, deep hollows, groove along dorsal margin (C. J. WEBB& M. J. A. SIMPSON,pets. comm.)

Cytology 26. Chromosome number: 2n = 22, 2n = 24, 2n = 48 (WANSCHER1933, SKOTTSBERG1955, RATTENBURY 1957, L6VE & RITCHIE 1966, SOKOLOVSKAYA1966, GRAHAM 1966, WARBLE 1968, STONE& LOO 1969, GURZENKOV1973 in GOLDBLATT1984, JAVURKOVA1981, L6VE & L6VE 1982, BEUZENBERG& HAIR 1983, STARODUBTSEV1985, SUN & al. 1988) Wood anatomy 27. Fibres: septate, not septate (METCALFE& CHALK 1950; SOPER 1957; CARLQUIST1981; OSKOLSKI 1994, 1995) 28. Vestured or warty layer: present, absent (OHTAN11979; OHTANI& al. 1983; BUTTERFIELD & al. 1984; OSKOLSKI1994, 1995) 29. Helical thickenings: present, absent (METCALFE& CHALK1950; MEYLAN& BUTTEREIELD 1974; CARLQUIST1981; BUTTERFIELD& al. 1984, OSKOLSKI1994, 1995; R. PATEL,pets. comm.) 30. Radial canals: present, absent (LEMESLE & DuPuY 1966; OSKOLSKI 1994, 1995; CARLQUIST1981) 31. Intervessel pitting: alternate, scalariform, opposite (BROWN 1922; METCALFE& CHALK 1950; RODRIGUEZ1957; SOPER1957; CARLQUIST1981; BUTTERFIELD& al. 1984; OSKOLSKI 1994, 1995) 32. Perforation plates: simple, scalariform with up to 10 bars, scalariform with more than 10 bars (BROWN 1922; SOPER 1957; MEYLAN & BUTTERFIELB 1978; CARLQUIST 1981; BUTTERFIELD& al. 1984; OSKOLSKI1994, 1995) 33. Ray types: homogeneous, heterogeneous, paedomorphic (METCALFE& CHALK 1950; SOPER 1957; CARLQUIST1981; OSKOLSKI1994, 1995) 34. Multiseriate ray sheath: absent, present (OSKOLSKI1994) 35. Vascular tracheids: absent, present (LEMESLE& DuPUY 1966; CARLQUIST1981; OSKOLSKI 1994, 1995)

option. The majority rule consensus tree (MARGUSH& MCMORRIS 1981) from combined data analysis was used as a representative of the most parsimonious trees. MacClade was used to map morphological apomorphies onto the majority rule tree.

128

A.D. MITCHELL& S. J. WAGSTAFF:

Results Molecular analysis. The data used in these analyses are available from the first author upon request, and the sequences have been submitted to GenBank (Table 1). It was possible to align all sequences generated, except those obtained from Stilbocarpa species. A total of 735 nucleotide positions were included in the analysis. The composition was of 22.1%A, 30.7%C, 29.1%G, 18.1%T, with 252-263 unambiguous transitions and 156-167 unambiguous transversions, calculated using the Resolve Polytomy option in MacClade. Ten equally parsimonious trees were found, 418 steps long, with consistency index (CI) 0.687 and rescaled consistency index (RC) 0.469. A strict consensus of the ten equally parsimonious trees is shown in Fig. 1. Astrotricha latifolia and A. ledifolia emerged at the base of the strict consensus tree and formed a monophyletic sister group to the other members of the Araliaceae. The remaining taxa were divided between two major monophyletic groups. The first of these groups consisted of 16 taxa, including Pseudopanax anomalus, P. edgerleyi, P. simplex, P. 9unnii, and P. davidii. Placement of P. gunnii within this large group was poorly resolved. The New Zealand P. anomalus, P. edgerleyi, and P. simplex (the Anomalus group) were monophyletic with Cheirodendron trigynum. Species from the Anomalus group were themselves monophyletic in eight of the ten equally most parsimonious trees. Pseuodopanax davidii emerged as the sister taxon to a group containing Eleutherococcus species and Kalopanax pictus. The second major monophyletic group consisted of 14 taxa, and included the Arboreus group (P. arboreus, P. colensoi, P. laetus, P. macintyrei, and P. kermadecensis) and the Crassifolius/Discolor group, comprising the Crassifolius (P. chathamicus, P. crassifolius, P. ferox, and P. linearis) and Discolor (P. lessonii, P. discolor, P. gilliesii) groups, plus Meryta sinclairii and M. pauciflora (Fig. 1). Twenty-two base insertions and 21 deletions were inferred (Table 3). Monophyly of the Arboreus group was supported by an inferred insertion of a C at position 125. The Crassifolius/Discolor group plus Arthrophyllum angustatum, Meryta sinclairii, and M. pauciflora shared a deletion of a C/T at position 237. The Anomalus group, plus P. davidii, Cheirodendron trigynum, Schefflera digitata, Fatsia japonica, and Tetrapanax papyriferus shared a deletion of a C at position 455. Pseudopanax arboreus, P. kermadecensis and P. macintyrei shared a deletion of an A/G at position 615. Morphological analyses. Taxa merged for analysis were P. arboreus, P. macintyrei, and P. colensoi; P. chathamicus and P. ferox; P. discolor and P. lessonii; and species from each of Astrotricha, Eleutherococcus, and Meryta. The heuristic search resulted in 270 equally parsimonious trees, each 234 steps, with CI--0.825 and RC = 0.580. Strict consensus of the 270 trees saved revealed several monophyletic groups, including the Anomalus, Arboreus and Crassifolius/Discolor groups of Pseudopanax (Fig. 1). Comparison between data sets. Molecular analysis supported the hypothesis that Pseudopanax is polyphyletic, but this was not possible to determine from morphology (Fig. 1). Both analyses showed three m a j o r Pseudopanax groups. In comparison with the molecular-based tree, the degree of resolution was less for the morphological tree. Trees were 42 steps longer when the molecular data set was


129

Table 3. Insertion and deletion events inferred as a result of alignment using manual pair-wise comparison Taxa

Pseudopanax lessonii Pseudopanax lessonii PoIysciasfi'uticosa Pseudopanax 9unnii Hedera helix Tetrapanax papyriferus Astrotricha latifolia, A. ledifolia Arthrophyllum angustatum Pseudopanax arboreus, P. colensoi, P. kermadecensis, P. laetus, P. macintyrei Astrotricha latifolia, A. ledifolia Aralia chinensis, Pittosporum dallii Astrotricha latifolia, A. ledifolia Aralia chinensis, Meryta sinclairii Pittosporum daIlii Pseudopanax 9unnii Pittosporum dallii Pseudopanax chathamicus, P. crassifolius, P. discolor, P. ferox, P. gilliesii, P. lessonii, P. linearis, ArthrophylIum angustatum, Meryta sinclairii, M. pauciflora Eleutherococcus nodiflorus Eleutherococcus nodiflorus Polysciasfruticosa Pseudopanax kermadecensis, P. laetus, Pittosporum dallii Polysciasfruticosa Eleu~herococcus nodiflorus Pittosporum daIIii Astrotricha latifolia, A. Iedifolia, Pittosporum dallii Pseudopanax anomalus, P. davidii, P. edgerIeyi, P. simplex, Cheirodendron trigynum, Schefflera digitata, Fatsia japonica, Tetrapanax papyriferus Pittosporum dallii Pittosporum dalIii Hedera helix Arthrophyllum angustatum Pseudopanax arboreus, P. kermadecensis, P. macintyrei Eleu~herococcus nodiflorus, Polyscias fruticosa Schefflera digitata Hedera helix Schefflera digitata

Insertions

Deletions

Position

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43 58 87-90 97 97 109 110 119 125

T,G,C,A T C G A/G/T C/A C A C C, C G G, C A T

C/T

C T/C C G A

128 192 193-194 195 196-197 236 236 237

240 271 385 418

C

422 441 453 454 455

A/G

471 476 494 523 615

C C/G C

A A A T

C/T C/T T, A C, A/C/G/T

637 650 662-663 664 665

analysed using the topology of the morphological strict consensus tree as a constraint. Trees were 16 steps longer when the morphological data set was analysed using the molecular strict consensus as a constraint. Analysis of the combined molecular and morphological data set when constrained to the topology of (1) the

130

A.D. MITCHELL¢~;S. J. WAGSTAFF: ITS-Strict

Morphology-Strict

Pseudqpanax anomalus Pseudbpanax edgerleyi rseuaopanax slmpte.x. rseuaopanax gunnn Pseudqpanax ~avidii Cheirodendron trigynum S~c,heffi.erGdig.itata rotysctas fruncosa Arthrophyllum angustatum Aralia cninensis Pseudopanaxarboreus~~ ~seudopanax kermadecensis Pseudopanax macintyre~ 1-'seuaop,anax cotensm n , rseuaopgn~ tae~us rseuaot~anax chamamicus Pseudo p a n ~ crassifolius rseuaopanax, jerox rseuqopanax unearts ~seuappanax alscoto.r. rseua_opanax tessonu Pseuddpanax gilliesii Eleutherococcus nodiflorus Eleutherococcus sessilifolius Hedera helix Kalopanaxpictus Tetrapanaxpapyriferus Fatsiajaponica Meryta sinclairii Meryta pauciflora I Astrotricha latifolia Astrotricha ledifolia Pittosporum dallii

Fig. 1. ITS-strict consensus of 10 equally most parsimonious trees, each 418 steps long, with consistency index (CI) 0.687 and rescaled consistency index (RC) 0.469, and morphologystrict consensus of 270 equally parsimonious trees, each 234 steps, with CI = 0.825 and RC = 0.580. Groups of New Zealand Pseudopanax species are highlighted

molecular strict consensus tree, and (2) the morphological strict consensus tree, resulted in trees respectively 1 step and 15 steps longer. Combined molecular and morphological analysis. Results of PAUP analysis using combined molecular plus morphological, anatomical, and cytological characters resulted in 18 equally parsimonious trees, 525 steps long, with CI -- 0.642 and RC = 0.4396. The strict consensus of these trees is shown as Fig. 2, and the majority rule tree is shown as Fig. 3. Of the 770 characters included in the analysis 137 were phylogenetically informative, and of these 105 were molecular and 32 morphological. Astrotricha latifolia and A. ledifolia formed a monophyletic group supported by 19 synapomorphies (bootstrap value 100%). Monophyly of the remaining members of the Araliaceae was supported by 12 synapomorphies (bootstrap value 50%). Next to diverge were the Meryta species, the monophyly of which was determined by 10 synapomorphies (bootstrap value 78 %) The remaining taxa were divided into two major monophyletic groups, the first supported by 6 synapomorphies and consisting of 16 taxa, including P. anomalus, P. edgerleyi, P. simplex, P. gunnii, and P. davidii (Fig. 3). Nine synapomorphies supported the relationship of P. gunnii with the monophyletic group containing P. anomalus, P. edgerleyi, P. simplex, and Cheirodendron trigynum (bootstrap value 58%). The monophyly of these New Zealand Pseudopanax with Cheirodendron trigynum was well supported by 6 synapomorphies (bootstrap value 80%).


131

Combinedanalysis-Strict

Pseudopanax anomalus

1

Pseudopanax si_mplex

j

Pseudopanax edgerleyi

58

Anomalus Group

Cheirodendron trigynum

Pseudopanax gunnii

501

:F

Pseu.do-panax ilavidii Eleutherococcus nodiflorus Kalopanax pictus Eleutherococcus sessilifoIius Hedera helix Tetrapanax papyriferus Fatsia japonica Schefflera digitata Aralia chinensis Arthrophyllum angustatum Polyscias fruticosa

89

Pseudopanax arboreus 7 Pseud_dpanax kermadecensisI Arboreus Pseudopanax macintyrei l Group rseua_opanax cotensm _J Pseudopanax laetus rseua_opanax clmOtamicus --[ Pseudopanax c rassifolius | Crassifolius rseuaopanaxj_erox Pseuddpanax linearis rseuaopanax aiscolor Pseuddpanaxgilliesii.

rseuaopanax tessonit

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100

I I

Meryta sinclairii Meryta pauciflora Astromcha latifolia Astrotricha ledifolia Pittosporum dallii

1 Group .3 -I

l

1

Discolor Group

Fig. 2. Strict consensus of 18 equally parsimonious trees found from analysis of combined ITS and morphological data set. Results of bootstrap analysis with 100 replications are given above the branches. Groups of New Zealand Pseudopanaxspecies are highlighted

Pseudopanax anomalus, P. edgerleyi, and P. simplex formed a monophyletic group defined by 7 synapomorphies (bootstrap value 100%), and the relationship of P. anomalus with P. simplex was also shown (bootstrap value 67%). Pseudopanax davidii, the Eleutherococcus species and Kalopanax pictus were monophyletic based on 9 synapomorphies (bootstrap value 72%). The second major monophyletic group containing Pseudopanax species was supported by 5 synapomorphies (Fig. 2). This large group contained two smaller monophyletic groups, the Arboreus group and the Crassifolius/Discolor group. Monophyly of the Arboreus group was supported by 12 synapomorphies (bootstrap value 100%) (Figs. 2, 3). The Crassifolius/Discolor group was supported by 8 synapomorphies (bootstrap value 89%) and included the smaller Crassifolius and Discolor groups. When topological constraints were imposed to reflect the monophyly of all Pseudopanax species, trees were 15 steps longer than the most parsimonious trees using the combined data. Similarly, forcing all New Zealand Pseudopanax species into monophyly produced trees that were 8 steps longer than the shortest trees. Discussion

There has been much debate over the use of heterogeneous data sets for phylogenetic analysis, especially morphology versus molecular data (e.g. HILLIS 1987, DONOGHUE & SANDERSON 1992, DE QUEIROZ & al. 1995). While researchers have combined different types of data prior to phylogenetic analysis (e.g. WI-mELER& al. 1993, KIM

132

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Fig. 3. Majority rule consensus tree generated from the combined ITS and morphology data set. Tree length was 525 steps with consistency index 0.642 and rescaled consistency index of 0.439. The branch length between Pittosporum dallii and the ingroup was 74 steps, and is not shown to scale. Non-molecular character support is shown for monophyletic groups. Thin bars with italicised numerals represent ambiguous synapomorphies, and thick bars with bolded numerals represent unambiguous synapomorphies. Characters are described in Table 2. Pseudopanax species are highlighted

& JANSEN 1994), independent analyses and estimation of taxonomic congruence was recommended by MIYAMOTO &; FITCH (1995). Analyses of both independent and combined data sets (SMITH & SYTSMA 1994) would appear to make best use of the available information. Comparison of trees obtained from ITS sequence data and morphology revealed several monophyletic groups c o m m o n to both, including the Pseudopanax Anomalus, Arboreus, and Crassifolius/Discolor groups. The low number of morphological characters available in relation to the number of taxa limited the analysis using this form of data alone. The combined molecular and morphological data analysis differed from the molecular analysis alone in the degree of resolution for Pseudopanax gunnii and Schefflera digitata, and in the position of the Meryta species (Figs. 1, 2). The phylogenetic separation of species in the Pseudopanax Anomalus group from the others endemic to New Zealand, and the isolated lineages ofP. 9unnii and P.


133

davidii clearly illustrate the polyphyly of Pseudopanax as it is presently circumscribed. Support for the monophyly of Pseudopanax species was lacking in the constraint analysis, when trees were substantially longer (15 steps) using the combined data. Pseudopanax species endemic to New Zealand form two distinct monophyletic groups, the Anomalus group and the Arboreus/Crassifolius-Discolor group (Fig. 2). The monophyly of these groups was not supported by constraint analysis, which resulted in trees 8 steps longer using combined data. While COCKAYNE& ALLAN(1934) and ALLAN(1961) suggested hybridisation between P. arboreus and P. simplex and between P. colensoi and P. simplex, more recent work (DP,ucE 1977: 88) has not accepted this. Pseudopanax anomalus, P. edgerleyi, and P. simplex (the Anomalus group) share a common ancestor with Cheirodendron trigynum and Pseudopanax 9unnii based on their coriaceous leaf texture, bilocular ovary and free style arms (Fig. 3). Information was lacking for the lateral face of the endocarp and the nodal anatomy of Cheirodendron trigynum and P. 9unnii, plus the wood anatomy of P. 9unnii. Monophyly of species from the Anomalus group (Fig. 2) is well supported by morphology in the combined data analysis (Fig. 3, Table 2), with synapomorphies including: the presence of unifoliolate adult leaves; juvenile leaves more complex than in the adult; inflorescence a compound raceme with three orders of branching (P. anomaIus has a uniquely derived simple umbel); axillary inflorescence (those of P. simplex are both axillary and terminal); and the presence of helical thickenings in the vessel elements of the wood. Helical thickenings are absent in the vessel elements of Cheirodendron trigynum subsp, helleri, C. 9audichaudii (= C. trigynum) and C. platyphyllum. Although helical thickenings occur infrequently in the AraliaceaeOSKOLSKI (1994) reported their presence in species of Astrotricha and Schefflera this study supports their multiple independent evolution in the family. Cheirodendron species form a clearly defined group based on the opposite leaf arrangement in adults, palmately compound leaf type, and the inflorescence: a terminal panicle of umbellules with four orders of branching. FP,ODIN (1990) reported the occurrence of alternate/spirally arranged leaves in reversion shoots of monocaulous juveniles of both C. trigynum subsp, helleri and C. platyphyllum, and this form of heteroblasty may represent a synapomorphy for the genus. There are five Cheirodendron species in Hawaii (LowRY 1990) and another in the Marquesas Islands (FRODIN 1990). A Pacific origin might be inferred for the Cheirodendron species and New Zealand's Pseudopanax Anomalus group. The hypothesised origin for these taxa is supported by FOS~ERG (1948), who suggested that the Hawaiian species of CHEIROD~NDRONmay have their origin in the South Pacific and have arisen from a single colonisation event. Pseudopanax davidii was found to be the sister taxon to the group containing Eleutherococcus species and Kalopanax pictus. Monophyly of this group was supported by ambiguous synapomorphies, including a bilocular ovary, style arms connate at the base and free at the tips, hermaphrodite sexuality, and absence of multiseriate ray sheath (Fig. 3). The close relationship of these taxa was also hypothesised by LI (1942). The combined molecular and morphological analysis indicates a close relationship between the remaining New Zealand Pseudopanax species from the Arboreus group and those from the Crassifolius/Discolor group (Figs. 2, 3). This relationship

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is supported by unambiguous synapomorphies, including coriaceous leaves and compound umbels (Fig. 3, Table 2). The majority of Pseudopanax species are contained in these groups, including the type for the genus, P. crassifolius. Hybrids form between P. arboreus and P. crassifolius (COCKAYNE& ALLAN 1934; METCALF 1972: 280--290), supporting the close relationship. Analysis of the combined data set indicates a more distant relationship between Pseudopanax and Meryta than the molecular analysis alone (Figs. 1, 2). The rbcL data of PLUNKETT & al. (1996) also supports a monophyletic Meryta (M. sinclairii) and Pseudopanax (P. arboreus). Endocarp morphology provides further character support for these taxa, i.e., the Crassifolius/Discolor group plus the Meryta species have grooves along the dorsal margin of the endocarp (C. J. WEB~ & M. J. A. SIMPSON,pers. comm.), a character not investigated for any Araliaceae outside New Zealand (Fig. 3, Table 2). A vestured or warty layer in the vessel elements of the wood (OHTANI& al. 1983, MEYLAN& BUTTERFIELD1974, BUTTERFIELD& al. 1984) supports the monophyly of the Pseudopanax Crassifolius/Discolor group. Natural hybrids exist between P. crassifolius and P. lessonii (COCKAYNE& ALLAN 1934), also supporting the close relationship. Although monophyly of the Crassifolius group was inferred from only 2 molecular synapomorphies, additional support comes from their simple adult leaf type and their thickly coriaceous leaf texture (SOPER1957, PHILIPSON1965). Molecular data does not clarify intra-group relationships for the Crassifolius group (Fig. 1). However, morphology suggests that P. chathamicus, P. crassifolius, and P. ferox share a more recent common ancestor than P. linearis, as determined by their completely fused style arms; P. linearis, P. discolor, P. 9illiesii and P. lessonii possess style arms that are connate at the base and free at the tips (PHILIPSON 1965). Monophyly of P. chathamicus, P. crassifolius, and P.ferox is also supported by the racemulate arrangement of their male flowers, although P. crassifolius may also have an umbellulate flower arrangement (PHILIPSON1965). Racemulate flower arrangement and wood anatomy supports monophyly of P. discolor, P. gilliesii, and P. lessonii, but relationships within the Discolor group are unresolved in the strict consensus tree (Fig. 3), indicating a close relationship and recent divergence for these taxa. Monophyly of the Arboreus group is supported by stipules (WARDLE1968), deep hollows in the lateral face of the endocarp (C. J. WEBB & M. J. A. SIMPSON,pers. comm.), and helical thickenings in the vessel elements of the wood (BUTTERFIELD & al. 1984; R. PATEL,pers. comm.). Ambiguous synapomorphies for the Arboreus group include their palmate primary leaf venation (PHILIPSON1965), 9 16 leaf traces in the petiole (SOPER 1957), bilocular ovary (PHILIPSON1965) and lack of a multiseriate ray sheath (investigated for P. colensoi, but not for other members of the group) (OSKOLSKI1994). The paucity of discrete morphological characters within the Arboreus group suggests recent divergence, and makes determination of withingroup relationships difficult. The two lineages found among the New Zealand Pseudopanax species, i.e. those leading to the Anomalus group and the Arboreus-Crassifolius/Discolor groups, may well be the result of independent origins. Alternatively many relatives of extant Araliaceae previously existed in New Zealand, and these taxa have since become extinct. Araliad pollen in New Zealand dates from the Upper Eocene (COUPER1960), around 50M years ago, to the present day. From mid Eocene to mid Oligocene times


135

the relative sea level rose around New Zealand, decreasing the land to 18% of its present area and increasing the rate of extinction (CooPER & MmLENER 1993). Similarly, many warm-temperate to subtropical plants became extinct during the severe cooling cycles of the Pleistocene (COOPER & MILLE~R 1993). Thus the hypothesis that many more Araliaceae may once have existed in New Zealand is not unrealistic. Future research. Increased sampling of Pacific Araliaceae and study of alternative sources of data may allow better evaluation of species relationships within the Arboreus group and the Crassifolius/Discolor group of Pseudopanax and in the genus Meryta. Inclusion of the Chinese Pseudopanax delavayi and P. rosthornii in the ITS data set is needed to test the monophyly of these taxa with P. davidii, and to determine their relationships with Eleutherococcus, and Kalopanax. According to D. G. F~ODIN (pers. comm.) Macropanax may be a close relative of P. davidii. This hypothesis, based on the form of the inflorescence, the evergreen habit, and the bilocular ovary (and fruit), could be tested within the ITS framework. Inclusion of the South American P. laetevirens would greatly add to possibilities for interpretation of biogeographical associations with New Zealand taxa, as would further research into the relationships of P. 9unnii from Tasmania. Pseudopanax gunnii is in need of morphological, anatomical, and cytological study towards a better understanding of its relationships. Pseudopanax classification will be re-evaluated in a separate paper. Particular attention will be given to species from the Anomalus group. Further research is being carried out in an attempt to determine close relatives of Stilbocarpa species. Many thanks to the staff and friends of herbarium CHR at Manaaki Whenua-Landcare Research, Lincoln, with special thanks to B. MACMILLANfor assistance and for requesting specimens. We thank C. J. WEBBand M. J. A. SIMPSONfor access to unpublished data from the seed atlas of New Zealand dicotyledons. R. PATEL assisted in the evaluation of wood anatomy characters and demonstrated the presence of helical thickenings in the vessel elements of Pseudopanax kermadecensis. We thanks H. WILSONfor help and assistance in locating populations of P. anomaIus and P. edgerleyi on Banks Peninsula. G. JORDAN (University of Tasmania) collected material of P. 9unnii, and W. SYKESand W. LEE(Landcare Research) provided material of Meryta pauciflora and Arthrophyllum angustatum respectively. B. RANCE(Department of Conservation, Invercargill) and G. SANTOS(Department of Conservation nursery, Motukarara) provided samples of Stilbocarpa species. Thanks to G. REDWOOD(Royal Botanic Gardens, Kew) and A. ROZEEELD(Tasmanian Herbarium, Hobart) for their support. Special thanks to D. FRODIN (Royal Botanic Gardens, Kew) and A. OSKOLSKI(Botanical Museum, St. Petersburg) for their help and advice on this project. We are grateful to the following herbaria for supplying specimens: ORSTOM, Noum~a, New Caledonia (NOU); Tasmanian Herbarium, Hobart, Tasmania (HO); ORSTOM de Tahiti, Papeete, Tahiti (PAP); MusEum National d'Histoire Naturelle, Paris, France (P); and Missouri Botanical Garden, Saint Louis, USA. (MO). References

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