Reconstructing the species phylogeny of Pseudopanax (Araliaceae), a genus of hybridising trees

Molecular Phylogenetics and Evolution 52 (2009) 774–783

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Reconstructing the species phylogeny of Pseudopanax (Araliaceae), a genus of hybridising trees Leon R. Perrie *, Lara D. Shepherd 1 Museum of New Zealand Te Papa Tongarewa, P.O. Box 467, Wellington, New Zealand

a r t i c l e

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Article history: Received 25 November 2008 Revised 19 April 2009 Accepted 28 May 2009 Available online 14 June 2009 Keywords: Pseudopanax Hybridisation, gene-flow, and introgression Phylogeny AFLP DNA-fingerprinting Chloroplast DNA sequences Araliaceae Neopanax Trees New Zealand

a b s t r a c t Pseudopanax (Araliaceae) comprises 12 tree species of diverse morphology and ecology, and is endemic to New Zealand. It is notable for the hybridisation that occurs between P. crassifolius and P. lessonii, which have very different leaves and habits. To provide context for the study of this hybridisation and other investigations, we examined the phylogeny of Pseudopanax using chloroplast DNA sequences (c.5900 base-pairs) and AFLP DNA-fingerprinting. Both approaches resolve two principal groups within Pseudopanax – the Arboreus group and the Crassifolius + Lessonii union – but how they are related to other genera remains unclear. There is, nevertheless, little compelling evidence against the monophyly of Pseudopanax, making unnecessary the recent re-segregation of the Arboreus group as Neopanax. The chloroplast data provided minimal additional resolution, although the position of P. linearis was discordant compared to other data. Analyses of the AFLP data strongly recovered each species, aside from the morphologically heterogeneous P. colensoi, and the two mainland species (P. arboreus and P. crassifolius) that each contained a nested island-endemic (P. kermadecensis and P. chathamicus, respectively). However, relationships amongst species within the two principal groups were poorly resolved. An example was the uncertainty of whether P. crassifolius grouped with P. lessonii and its allies, or the morphologically similar species it had been previously placed with. This in turn raises the issue of how hybridisation might affect phylogenies and the ability to reconstruct them, even when using multiple, independent markers. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction An understanding of phylogeny, or how species are related to one another, facilitates a more comprehensive interpretation of evolutionary processes, including speciation and hybridisation (Harrison, 1998). The latter two themes are intertwined because it is believed that the study of hybridising species can reveal the factors maintaining species’ boundaries, which in turn may be informative as to what is initially important in speciation (Harrison, 1993; Lexer et al., 2005). Recent studies indicate that significant hybridisation and introgression following speciation may be common, and that the genetic basis for isolation may be very small, with much introgression occurring amongst parts of the genome not tightly linked to these isolating genetic elements (Hey, 2006; Yatabe et al., 2007). It is widely recognised (e.g., Rieseberg and Soltis, 1991) that introgression can lead to discordance between the phylogeny inferred from a single locus (i.e., a ‘gene-tree’) and that inferred from multiple independent characters (i.e., the ‘species-

* Corresponding author. Fax: +64 4 381 7070. E-mail address: [email protected] (L.R. Perrie). 1 Present address: Allan Wilson Centre, Massey University, Palmerston North, New Zealand. 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.05.030

tree’). Consequently, phylogenies are being increasingly reconstructed using multi-locus genetic markers, but there has been little exploration of how such phylogenetic inferences might be affected by introgression, and, in turn, what this means for using them to interpret hybrid zones. Furthermore, studies of hybridisation involving species with a broader range of demographic, ecological, and reproductive characteristics are required before patterns and processes can be widely generalised. A striking example of hybridisation occurs in Pseudopanax (Araliaceae). The circumscription of this genus is historically complex, and still contentious (see below). Based largely on the results of Mitchell and Wagstaff (1997, 2000), we regard Pseudopanax as comprising the species listed by Philipson (1965), but with the addition of P. macintyrei (Wardle, 1968), and the exclusion of non-New Zealand species and the species subsequently referred to Raukaua (Mitchell et al., 1997). So defined, Pseudopanax is endemic to New Zealand and constitutes 12 small trees or large shrubs, that are dioecious (Webb et al., 1999), and range from coastal to subalpine habitats. Extraordinary hybridisation occurs between P. crassifolius (lancewood, horoeka) and P. lessonii (coastal five-finger, houpara), in that (1) their leaves (Fig. 1) and habit (see below) are very different; (2) their hybrids are morphologically diverse (see Fig. 1 for examples of hybrids’ leaves); (3) hybridisation occurs at

L.R. Perrie, L.D. Shepherd / Molecular Phylogenetics and Evolution 52 (2009) 774–783

Fig. 1. Leaves of adult (left-most) and juvenile (second from left) Pseudopanax crassifolius and P. lessonii (right-most). The other leaves are from wild P. crassifolius  lessonii plants.

many sites (Fig. 2); and (4) hybrids are abundant at many of these sites. The genetic constitutions of these hybrids are unknown, as is the extent of introgression between the parental species. However, unpublished data indicate that hybrids can produce viable fruit (Pollock, 1988). Pseudopanax crassifolius is a forest-successionist, found from low to montane altitudes throughout the North, South, and Stewart Islands (Fig. 2). The indigenous distribution of P. lessonii is restricted to coastal forests of the northern North Island (Fig. 2). Hybridisation, with the production of morphologically-diverse swarms, commonly occurs throughout the indigenous distribution of P. lessonii (Fig. 2), as well as in many other regions where it has been cultivated as a garden ornamental in close proximity to P. crassifolius. It appears that Pseudopanax crassifolius and P. lessonii are not even sister species. Pseudopanax lessonii is morphologically closer to the similarly multi-foliolate P. discolor and P. gilliesii, both of which have narrow distributions in the northern North Island. This Lessonii group was recovered by Mitchell and Wagstaff (1997) with 81% bootstrap support (BS) in a maximum parsimony analysis of a data set comprising ITS DNA sequences and morphological characters. Like Pseudopanax crassifolius, P. chathamicus, P. ferox, and P. linearis all have simple leaves. Both P. crassifolius and P. ferox, which occurs sparsely throughout the lowlands of the North and South Islands, exhibit striking leaf (Fig. 1) and habit heteroblasty between juveniles and adults. Heteroblasty is a noted feature of the New Zealand flora (Burns, 2005; Burns and Dawson, 2006), occurring in many unrelated trees and shrubs. The reason for its prominence remains controversial, being variously attributed to biotic (e.g., browsing by the now extinct moa ratites) or abiotic factors. Heteroblasty is less pronounced in P. linearis, a montane to subalpine

Fig. 2. Maps of the indigenous distributions of (A) Pseudopanax crassifolius, (B) P. lessonii, and (C) P. crassifolius  lessonii. Compiled using records from the AK, CHR, and WELT herbaria, plus the authors’ own collections.

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species of the South Island, and only weakly expressed in the Chatham Islands’ endemic P. chathamicus. This Crassifolius group was recovered with 75% bootstrap support by Mitchell and Wagstaff (1997). Based on overlapping distributions, it is possible that other hybrid combinations occur (e.g., P. discolor, P. ferox, or P. gilliesii with P. lessonii or P. crassifolius; P. linearis with P. crassifolius), but none appear to occur as frequently as P. crassifolius  lessonii. The remaining Pseudopanax species have multi-foliolate and stipulate leaves, and belong to the Arboreus group: P. arboreus is a lowland to montane species found throughout the North Island and extending to the north and east of the South Island; P. colensoi, in which three varieties have been recognised (Wardle, 1968), is a montane to subalpine species of the North, South, and Stewart Islands; P. laetus occurs sparsely in the central North Island; P. macintyrei is restricted to the northwest of the South Island; and P. kermadecensis is endemic to the Kermadec Islands. Some hybridisation may occur within the Arboreus group (e.g., P. colensoi with P. arboreus and P. macintyrei), but there is little evidence for hybridisation between the Arboreus group and either of the Crassifolius or Lessonii groups (but see Cockanye and Allan, 1926; Metcalf, 2000). Although the relationship between the Crassifolius and the Lessonii groups is close (Mitchell and Wagstaff, 1997), the Arboreus group may be distantly related. Indeed, as defined here, Pseudopanax may not be monophyletic because of the possible interpolation of Meryta and the so-called ‘‘Pacific” subgroup of the polyphyletic Schefflera (Plunkett et al., 2004, 2005; Tronchet et al., 2005). The simple-leaved and largely south Pacific Meryta, with about 30 species, and the palmately-compound-leaved Pacific Schefflera subgroup, with some 25+ species, appear to form a clade, but it is not clear how Pseudopanax is related to them. For instance, Mitchell and Wagstaff (1997) found Meryta to be sister to the Crassifolius + Lessonii clade with ITS DNA sequences (bootstrap support not reported). They recovered a monophyletic Pseudopanax with a combined ITS and morphological analysis (they did not sample Pacific Schefflera). In contrast, Tronchet et al. (2005) found the Arboreus group to be sister to the Meryta + Pacific Schefflera clade with ITS, although this was very poorly supported (46% bootstrap support with maximum parsimony). Plunkett et al. (2004) reported, from a combined analysis of ITS and chloroplast trnL–trnF DNA sequences, only a polytomy between the Meryta + Pacific Schefflera clade, the Crassifolius + Lessonii clade, and the Arboreus group. Frodin and Govaerts (2004) stressed the differences between the Crassifolius + Lessonii group and the Arboreus group. They reinstated the genus Neopanax for the species of the Arboreus group, but we retain them here in Pseudopanax (see Section 4). The only previous phylogenetic study to include all of the Pseudopanax species employed only a single sample per species, and found little ITS sequence variation so that some species were not distinguished and many relationships were not well-supported (Mitchell and Wagstaff, 1997). Aside from the taxonomic issue of generic boundaries, clarifying the relationships amongst the species of Pseudopanax is of great interest because the morphological and ecological diversity in such a small group makes it an appealing choice for examining factors promoting speciation, morphological/ecological diversification (including heteroblasty), and hybridisation, as well as phylogeographic investigations of where New Zealand’s forests survived during the Pleistocene (e.g., Shepherd et al., 2007). We have an ongoing project (e.g., Shepherd et al., 2008) including these topics that requires a more detailed phylogenetic framework than presently available for the genus. Therefore, in order to better understand its phylogeny, we used AFLP DNA-fingerprinting (Vos et al., 1995) to examine Pseudopanax. Despite its limitations (Koopman, 2005), AFLP has been described as the method ‘‘of choice to reconstruct species relationships in evolutionary complex groups”, such as those experiencing hybridisation, because it assesses numerous

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markers from throughout the genome (Koopman et al., 2008). We also generated DNA sequences from multiple chloroplast loci to test their utility both for phylogenetic reconstruction and, because of their likely maternal inheritance, for assessing the parental symmetry of the hybridisation (Arnold, 1993) between P. crassifolius and P. lessonii.

error rates. Duplicates were discarded prior to analyses. Automated scoring was also conducted, with a range of 50–500 basepair (bp), peak detection threshold intensity of 100, the stutter peak and plus-A filters on, and with peak score values of reject = 1 and pass = 7, to assess whether this affected the major inferences made from analyses of the manually-scored data. 2.2. Analyses of AFLP data

2. Methods 2.1. AFLP data generation AFLP profiles were produced for multiple specimens from each Pseudopanax species, with multiple populations sampled for those species with wide distributions (Table 1). More samples were analysed for P. colensoi because of its morphological and taxonomic variation (Wardle, 1968), and for P. crassifolius and P. lessonii because of our ongoing interest in their hybridisation. However, so that any underlying phylogenetic signal amongst the species was not obscured by hybrids, only samples with no obvious indication of hybridisation/introgression were included, as judged from their morphology and/or preliminary genetic analyses. Additionally, single samples were processed for Meryta sinclairii, M. latifolia, Raukaua anomalus, and Fatsia japonica, as potential outgroups. Meryta is closely related to Pseudopanax, but Raukaua and Fatsia much less so (Plunkett et al., 2004). Genomic DNA was extracted from fresh leaves using a modified CTAB method (Doyle and Doyle, 1990). For each sample, approximately 200 ng of DNA was restricted with 5 U of EcoRI (Roche), 5 U of MseI (NEB), 0.625 lmol KOAc, 0.125 lmol MgOAc, and 0.125 lmol Tris–HCl (pH 7.5), in a total volume of 12.5 lL, at 37 °C for 3 h, followed by inactivation of the enzymes at 70 °C for 15 min. 5 lL of the restricted DNA was electrophoresed on 1% agarose to confirm that the digestion was complete. Linkers for the AFLP PCR were ligated to another 5 lL of the restriction digestion using 0.5 lL of each of the Eco and Mse linkers (see Vos et al., 1995), 0.5 U of T4 DNA ligase (Roche), and 1 ligation buffer (Roche), in a total volume of 10 lL, and incubated overnight at 4 °C. Pre-amplification PCR reactions contained 20 lmol Betaine, 5 nmol dNTPs (Roche), 1 PCR buffer (BioLine), 30 nmol MgCl2, 10 pmol Eco-A primer, 10 pmol Mse-C primer, 1 U Taq polymerase (BioLine), and 1 lL ligated DNA, in a total of 20 lL. Pre-amplification thermocycling conditions comprised 20 cycles of 94 °C for 30 s, 56 °C for 1 min, and 72 °C for 1 min, with a ramping of 1 °C/ s for all steps. Selective PCR amplifications contained 5 nmol dNTPs (Roche), 1 PCR buffer (BioLine), 92.5 nmol MgCl2, 10 pmol of each of the Eco and Mse selective-primers, 1 U Taq polymerase (BioLine), and 1 lL of the pre-amplification product, in a total of 20 lL. Four selective primer combinations were chosen from a primer screen: 6Fam–Eco–ACT with Mse–CTG, Vic–Eco–ATA with Mse–CCC, Ned–Eco–ACC with Mse–CGT, and Pet–Eco–AGG with Mse–CAA (Sigma, except ABI for the Vic, Ned, and Pet labelled primers). Selective thermocycling conditions comprised an initial denaturation step of 94°C for 2 min, 10 cycles of 94 °C for 30 s, 65 °C for 30 s ( 1 °C per cycle), and 72 °C for 1 min, 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, and a final extension of 72 °C for 30 min; ramping was set to 1 °C/s for all steps. Selective PCR products were pooled in equal proportions, with 0.9 lL of this mixture processed alongside 0.1 lL of a Liz-labelled size standard on an ABI3730 capillary sequencer (Allan Wilson Centre Genome Service, Palmerston North, New Zealand). The AFLP profiles were scored manually using GeneMarker v.1.71 (SoftGenetics) to produce a binary matrix (0/1), with uncertainty scored as ‘?’. Profiles with low signal and/or number of characters scored as 1 were discarded. Fourteen samples were processed in duplicate beginning at the restriction stage to gauge

Alternative approaches of analysing AFLP data can result in quite different conclusions (e.g., Albach, 2007). Therefore, the Pseudopanax AFLP data were assessed using a number of treebuilding methods, to investigate which inferences of relationships were robust across different approaches. Neighbour-joining was taken to provide a reasonable representation of phylogenetic relationships (see Spooner et al., 2005; Tremetsberger et al., 2006), alongside parsimony and Bayesian analyses. The latter approach is being increasingly applied to AFLP data (e.g., Albach, 2007; Koopman et al., 2008). The terms monophyletic (plus sister) and paraphyletic were used to describe the inferred relationships amongst the samples regardless of the methodology used, and with the assumption that the root was outside either of the Crassifolius + Lessonii and Arboreus groups (i.e., one is not nested within the other). This was supported by mid-point rooting of the AFLP data (see Fig. 3) as well as analyses of the chloroplast DNA sequences (see Section 3). It is expected that these tree-building methods may provide poor representations of infra-specific relationships that are non-bifurcating because of gene-flow. However, these tree-building methods should detect – as branches consistently recovered with high support – the boundaries between divergently related groups where lineage-sorting has engendered concordant partitioning across multiple characters (Perrie et al., 2003a), as well as the relationships between such groups. It is these supra-lineage relationships that we are principally interested in here. The samples of Fatsia and Raukaua were removed prior to the analyses described below, as their AFLP profiles were very different to those of Pseudopanax and Mertya, and they are likely to be too distant for their relationships to the remainder of the samples to be recovered reliably (Koopman, 2005). The Meryta samples were retained, but they were not used as outgroups because it is unclear whether Pseudopanax is monophyletic (see Section 1). Neighbour-joining (NJ) trees were constructed using PAUP 4.0b10 (Swofford, 2002), based on total character (TC-NJ) and Nei-Li (NL-NJ) distances. A maximum parsimony (MP) analysis was also conducted using PAUP, with a heuristic search of 1000 random addition replicates and TBR branch-swapping. Branch support was assessed using 1000 bootstrap pseudoreplicates for NJ and MP, with 10 random addition replicates for each MP pseudoreplicate. A Bayesian analysis (BA) was carried out using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001), with a binary model (nst = 1), coding = noabsencesites (see Koopman et al., 2008), and default priors. Two concurrent analyses were run, each with four Markov chains of 5,000,000 generations. These were sampled every 1000 generations, and the first 50% of these samples were discarded as ‘burn-in’. At this point, the standard deviation of split frequencies was less than 0.01, indicating convergence to a stationary distribution had been achieved. To examine consistency across the tree-building methods, a consensus network was constructed from the TC-NJ, NL-NJ, 50% majority rule (plus other compatible groups) of the MP trees, and the 50% majority rule from the BA, using SplitsTree 4.10 (Huson and Bryant, 2006) with a threshold of 0.2. Additional analyses were carried out to investigate whether the recovered relationships amongst the Crassifolius and Lessonii groups were being affected by introgression, particularly from hybridisation between Pseudopanax crassifolius and P. lessonii. The

L.R. Perrie, L.D. Shepherd / Molecular Phylogenetics and Evolution 52 (2009) 774–783 Table 1 Samples included in the AFLP and/or chloroplast DNA sequence analyses. Samples are numbered by their WELT herbarium registrations (other than the two samples accessioned at AK). For the samples included in the nine-species set, GenBank accession numbers are given in the order: trnfM–trnS region, rbcL gene, rps4 region, psbA–trnH spacer, psbA–trnK spacer, trnT–trnE spacer, and rpL16 intron. GenBank accession numbers for just the first three loci are listed for the additional samples included in the 18-species set. Sample

Locality

Pseudopanax arboreus (Murray) Philipson SP86457 Puketi Forest SP86446 Mount Taranaki SP86467 East Cape SP86506 Wellington SP86438 SP86494

GenBank accession numbers

FJ470202, FJ470142, FJ470235

FJ470201, FJ470141, FJ470234, FJ470158, FJ470167, FJ470176, FJ470251

Kaikoura Banks Peninsula

P. chathamicus Kirk SP86466 Chatham Island AK300993 Chatham Island AK301274 Pitt Island SP86425 Chatham Island P. colensoi (Hook.f.) Philipson var.colensoi SP86431 Pirongia Mountain SP86447 Mount Taranaki SP86453 Central Plateau SP86460 Banks Peninsula

P. crassifolius (A.Cunn.) K.Koch SP86470 Puketi Forest SP86503 Auckland SP86468 East Cape SP86427 Ruahine Ranges SP86477 SP86479 SP86493 SP86437 SP86483 SP86490

Wellington Denniston Banks Peninsula Mount Cook Te Anau Catlins

P. discolor (Kirk) Harms SP86496 Great Barrier Island SP86435 Coromandel Peninsula SP86449 Coromandel Peninsula SP86452 Coromandel Peninsula SP86478 Coromandel Peninsula P. ferox Kirk SP86469 SP86476 SP86495 SP86492 SP86489 SP86430

Auckland Rimutaka Ranges Banks Peninsula Dunedin Invercargill Cultivated

Sample

Locality

P. gilliesii Kirk SP86426 Whangaroa Harbour SP86474 Whangaroa Harbour SP86472 Whangaroa Harbour SP86455 Kaeo SP86456 Kaeo P. kermadecensis (W.R.B.Oliv.) Philipson SP86439 Raoul Island SP86440 Raoul Island SP86441 Raoul Island P. laetus (Kirk) Philipson SP86451 Coromandel Peninsula SP86444 Kawhia a Kawhia SP86513

FJ470193, FJ470133, FJ470226

FJ470197, FJ470137, FJ470230 FJ470194, FJ470134, FJ470227

P. colensoi var. fiordensis Wardle Bluff 1 FJ470207, FJ470147, FJ470240 SP86485a SP86486 Bluff 1 SP86487 Bluff 2 SP86488 Bluff 2 P. colensoi var. ternatus Wardle SP86445 St. Arnaud SP86464 Denniston SP86479 Ross SP86436 Mount Cook SP86491 Catlins SP86484 Te Waewae Bay

Table 1 (continued)

SP86475 SP86429

National Park Cultivated

P. lessonii (DC.) K.Koch SP86497 North Cape SP86458 North Cape SP86459 North Cape SP86474 Cape Maria van Diemen SP86428 Karikari Peninsula SP86473 Whangaroa SP86443 Whangarei SP86454 Whangarei SP86432 Auckland

FJ470196, FJ470136, FJ470229

SP86450

FJ470195, FJ470135, FJ470228

SP86448 SP86442 SP86433

GenBank accession numbers

FJ470189, FJ470129, FJ470222

FJ470211, FJ470151, FJ470244

FJ470199, FJ470139, FJ470232, FJ470160, FJ470169, FJ470178, FJ470253 FJ470198, FJ470138, FJ470231

FJ470188, FJ470128, FJ470221

FJ470185, FJ470125, FJ470218

FJ470184, FJ470124, FJ470217, FJ470157, FJ470166, FJ470175, FJ470250

Coromandel Peninsula Waihi Whakatane East Cape

P. linearis (Hook.f.) K.Koch SP86434 Heaphy Track FJ470187, FJ470127, FJ470220 Denniston Arthur’s Pass Haast Pass

FJ470191, FJ470131, FJ470224, FJ470159, FJ470168, FJ470177, FJ470252 FJ470192, FJ470132, FJ470225

FJ470183, FJ470123, FJ470216, FJ470156, FJ470165, FJ470174, FJ470249

SP86465 SP86480 SP86482

FJ470186, FJ470126, FJ470219

P. macintyrei (Cheeseman) Wardle SP86461 Takaka Hill FJ470200, FJ470140, FJ470233 SP86462 Takaka Hill SP86463 Murchison Meryta denhamii Seem. Cultivated SP86500a

FJ470205, FJ470145, FJ470238

M. latifolia Seem. SP86501 Cultivated

FJ470206, FJ470146, FJ470239

FJ470204, FJ470144, FJ470237, FJ470154, FJ470163, FJ470172, FJ470247 FJ470208, FJ470148, FJ470241

M. sinclairii (Hook.f.) Seem. SP86499 Cultivated

FJ470203, FJ470143, FJ470236, FJ470155, FJ470164, FJ470173, FJ470248 Fatsia japonica (Thunb.) Decne. et Planch. Cultivated FJ470179, FJ470119, FJ470212 SP86502b Raukaua anomalus (Hook.) A.D.Mitch., Frodin, et Heads SP86505b Dannevirke FJ470180, FJ470120, FJ470213, FJ470152, FJ470161, FJ470170, FJ470245 FJ470209, FJ470149, FJ470242 FJ470210, FJ470150, FJ470243 FJ470190, FJ470130, FJ470223

Schefflera digitata J.R.Forst. et G.Forst. Ruahine Ranges FJ470181, FJ470121, FJ470214, FJ470153, SP86504a FJ470162, FJ470171, FJ470246 a b

AFLP profile not generated. AFLP profile generated and scored, but not included in AFLP analyses.

777

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Fig. 3. Neighbour-joining tree using Nei-Li distances (NL-NJ) of the Pseudopanax AFLP data. The tree is mid-point rooted. Branches with P80% BS and/or 0.95 PP in at least three of the four tree-building methods are thickened. See Table 1 for sample details.

presence of conflicting signals in the AFLP data was assessed using SplitsTree to construct a neighbour-net (ordinary least squares variance, and threshold = 0.001). Structure 2.2 (Falush et al., 2007) was used to examine the samples of the Crassifolius and Lessonii groups (using only the characters variable amongst this set), with the admixture model, correlated allele frequencies, 100,000 burnin generations, a further 100,000 generations for sampling, and K = 2. Finally, the effect of excluding various sets of P. crassifolius and/or P. lessonii samples from the NJ and BA analyses was tested. Of particular interest in P. crassifolius were the three northern samples – WELT SP86503, SP86468, and SP86470 (Table 1) – that occur within the indigenous distribution of P. lessonii. 2.3. Chloroplast DNA sequences Chloroplast DNA (cpDNA) sequences were generated for a ninespecies set with a focus on finding loci that would be useful for differentiating Pseudopanax lessonii and P. crassifolius and/or reassessing the relationship of Pseudopanax and Meryta. Ten amplicons were initially investigated in one sample of each of P. lessonii, P. crassifolius, P. arboreus, and the outgroup Raukaua anomalus (Table 1). These were the rps4 region (rps4 gene + rps4–trnS spacer); psbA–trnH spacer; psbA–trnK spacer; rbcL gene; trnT–trnE spacer; rpL16 intron; trnfM–trnS region (trnfM–trnG spacer + trnGGCC gen-

e + trnG–ycf9 spacer + ycf9 gene + ycf9–trnS spacer); petN–psbM spacer; trnL–trnF region (trnL intron + trnL–trnF spacer); and rps16 intron; see Supplementary Table 1 for primers and relevant references). The first seven amplicons sequenced cleanly and exhibited differences amongst the above Pseudopanax species, so additional sequences were generated for these amplicons from single samples of P. linearis, P. laetus, Meryta sinclairii, M. denhamii, and a further outgroup Schefflera digitata (the nine-species set; Table 1). PCR amplification was performed in 20 ll volumes containing 1 PCR buffer (10 mM Tris–HCl, 50 mM KCl, pH 8.3; Roche Applied Science, Auckland), 1.5 mM MgCl2, 1 M betaine, 250 lmol dNTPs, 10 qmol of each primer, 1U Taq polymerase (Bioline), and approximately 0.5–3 ng of template DNA. PCR thermocycling conditions involved an initial denaturation step of 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 50–57.5 °C (see Supplementary Material) for 1 min, and 72 °C for 1.5 min, and a final extension of 72 °C for 5 min. PCR products were purified by digestion with 1 U shrimp alkaline phosphatase (SAP, USB Corp., Cleveland, USA) and 5 U exonuclease I (Exo I, USB Corp., Cleveland, USA) at 37 °C for 30 min, followed by inactivation of the enzymes at 80 °C for 15 min. The purified PCR products were then used directly for DNA sequencing. All PCR products were sequenced in both directions with the ABI Prism Big Dye Terminator cycle sequencing kit version 3.1 and

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run on an ABI 3730 DNA sequencer (Allan Wilson Centre Genome Service, Palmerston North, New Zealand). The three most informative loci – rps4 region; rbcL gene; and trnS–trnfM region – were sequenced as described above for all of the Pseudopanax species to investigate their chloroplasts’ relationships. This 18-species set comprised five samples of P. colensoi, three samples for each of P. crassifolius, P. ferox, P. lessonii, and P. linearis, two samples for each of P. arboreus and P. laetus, and single samples for each of the five remaining Pseudopanax species as well as Meryta sinclairii, M. denhamii, M. latifolia, and the outgroups Fatsia japonica, Raukaua anomalus, and Schefflera digitata (a total of 32 samples; Table 1). These latter three Araliaceae species are all clearly outside the clade that includes Pseudopanax and Meryta (Plunkett et al., 2004). The sequences for each locus were aligned by eye. Regions that could not be unambiguously aligned were omitted. For the two different sample sets, the relevant loci were concatenated, following pairwise partition homogeneity tests in PAUP with invariant sites removed (Farris et al., 1994). Redundant concatenated sequences were omitted before analyses. Insertion/deletion (indels) events were treated as missing data, but MP for the nine-species set was repeated with unambiguous and informative indels recoded as binary characters. Phylogenies for each concatenated taxa set were constructed in PAUP using maximum parsimony (MP) and maximum likelihood (ML). A heuristic search algorithm with 100 random addition sequence replicates and TBR branch-swapping was used for both methods. For ML, the most appropriate model of sequence evolution for each species set (nine-species set: TrN + I + C; base frequencies of A = 0.3341, C = 0.1790, G = 0.1806, T = 0.3063; rate matrix = [1.0000, 1.4266, 1.0000, 1.0000, 0.8404]; I = 0.8309; C = 0.8776. 18-species set: TVM + I; base frequencies of A = 0.2965, C = 0.1910, G = 0.2044, T = 0.3081; rate matrix = [1.4568, 1.8695, 0.4425, 1.2116, 1.8695]; I = 0.9054) was determined using the Akaike information criterion (AIC) in Modeltest 3.7 (Posada and Buckley, 2004). Nodal support was assessed by 100 (ML) or 1000 (MP) bootstrap replicates (BS). Bayesian analyses

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(BA) were also performed, using MrBayes with substitution parameters unlinked between the amplicons, nst = 6, rates = invgamma, and the default priors. Two concurrent analyses were run, each with four Markov chains of 10,000,000 generations. The chains were sampled every 1000 generations, and the first 50% of these samples were discarded as ‘burn-in’. At this point, the standard deviation of split frequencies was less than 0.01, indicating convergence to a stationary distribution had been achieved. The Shimodaira–Hasegawa test (SH; Shimodaira and Hasegawa, 1999), as implemented in PAUP, was performed on the nine-species set to assess competing topologies, specifically in regard to whether Pseudopanax crassifolius was more closely related to P. lessonii or P. linearis. The SH test was run with 1000 RELL bootstrap replicates. The SH test was only conducted for the nine-species set, since this had sequence data for more loci, and therefore assumedly afforded the most power. 3. Results 3.1. AFLP The final AFLP data set for Pseudopanax and Meryta comprised 78 samples, with 911 AFLP characters. Eight hundred and ninetyfive of these were polymorphic, and 745 were parsimony informative. The TC-distance error rate from the additional 14 duplicate samples ranged from 0.9 to 5.7%, with a median of 2.2%, in line with previous findings of a high reproducibility for AFLP profiles (Bonin et al., 2004). The NL-NJ is shown in Fig. 3. Trees from the other analyses are not shown, but the consensus network for all four tree-building methods, representing all of the edges they recovered, is shown in Fig. 4. Some relationships were recovered across all of the tree-building methods, and are shown as non-boxed edges in the consensus network; many of these same relationships were also strongly supported. The MP analysis recovered 229 trees of score 3924, across 23 islands.

Fig. 4. Consensus network of the TC-NJ, NL-NJ, 50% consensus MP, and 50% consensus BA trees. Lengths of edges are proportional to how many of the four source trees that edge occurred in. Boxes indicate conflict between the trees from the different methods. See Table 1 for sample details.

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The samples of Pseudopanax chathamicus, P. discolor, P. ferox, P. gilliesii, P. kermadecensis, P. lessonii, P. linearis, P. laetus, and P. macintyrei were each recovered as ‘monophyletic’ with 100% TC-NJ, 100% NL-NJ, and 100% MP BS, 1.0 PP, and in 100% of the MP trees; except P. chathamicus had 99% TC-NJ, 99% NL-NJ, and 98% MP BS, P. discolor had 94% MP BS, P. ferox had 99% TC-NJ, 96% NL-NJ, and 97% MP BS, and P. lessonii had 98% TC-NJ and 92% MP BS. Pseudopanax chathamicus was nested within P. crassifolius, their union (hereafter, P. crassifolius + chathamicus) receiving 99% TC-NJ, 99% NL-NJ, and 99% MP BS, 0.97 PP, and being found in 100% of the MP trees. Similarly, P. kermadecensis was nested within P. arboreus, their union (hereafter, P. arboreus + kermadecensis) receiving 90% TC-NJ, 89% NL-NJ, and 77% MP BS, 0.88 PP, and being found in 100% of the MP trees. Both P. crassifolius and P. arboreus might be better regarded as ‘metaphyletic’ or unresolved (Rieseberg and Brouillet, 1994) rather than ‘paraphyletic’, since few of the relevant edges had support greater than 80% BS or 0.90 PP (except for an edge with 96% TC-NJ BS within P. arboreus and one with 1.0 PP within P. crassifolius). Pseudopanax colensoi var. fiordensis was strongly supported (100% TC-NJ, 100% NL-NJ, and 98% MP BS, 1.0 PP, and in 100% of the MP trees), but P. colensoi var. colensoi, P. colensoi var. ternatus, and P. colensoi as a whole were not; indeed, the latter two taxa were not even consistently monophyletic. The Arboreus group, the Lessonii group, and the union of the Crassifolius and Lessonii groups (hereafter, the Crassifolius + Lessonii union) were each strongly supported (all 100% BS, 1.0 PP, 100% of the MP trees, except 92% MP BS for the Lessonii group). The Crassifolius group was only recovered as monophyletic in the MP (100% of the MP trees, although it had <50% BS). In the other analyses, the Lessonii group was nested within the Crassifolius group, with Pseudopanax crassifolius + chathamicus sister to the Lessonii group (68% TC-NJ and 66% NL-NJ BS, 0.67 PP). The 0.95 posterior tree set of the BA was dominated by trees with P. crassifolius + chathamicus sister to the Lessonii group (3132 of the 4627 total trees), but trees with a monophyletic Crassifolius group sister to a monophyletic Lessonii group were also present (670 trees). Pseudopanax ferox was variously sister to P. linearis (<50% NL-NJ and 59% MP BS, 0.73 PP) or to P. linearis plus the remainder of the combined Crassifolius and Lessonii groups (53% TC-NJ BS). Within the Lessonii group, P. gilliesii was either sister to P. lessonii (61% TC-NJ and 52% MP BS, 93% of the MP trees) or to P. discolor (55% NL-NJ BS and 0.68 PP). The relationships amongst the species of the Arboreus group were even less clear (Fig. 4), with nothing, other than the P. arboreus + kermadecensis union (see above), receiving BS > 55% or PP > 0.59. The neighbour-net from SplitsTree was quite ‘boxy’ (not shown); few, if any, of the internal edges were without conflict, which probably reflected the spanning of infra- and inter-specific relationships in this sample set and the general noisy nature of AFLP data. Nevertheless, three principal groups were evident, corresponding to Meryta, the Arboreus group, and the Crassifolius + Lessonii union, and the samples of each species were grouped together (although not always with an edge corresponding exactly to the species). If pronounced introgression was occurring, it might be expected that an edge splitting some of the Pseudopanax crassifolius samples (particularly those from the north) with some or all of the P. lessonii samples from the remaining samples would be present, but this was not the case. The Structure analysis strongly distinguished the Crassifolius group from the Lessonii group, with no indication of substantial introgression between Pseudopanax crassifolius and P. lessonii, as judged with a sample set from which putative hybrids had been excluded. The only samples with Q values less than 0.95 were the four P. linearis samples (Q  0.85 to the Crassifolius group) and two samples of discolor (SP86452; Q = 0.94 to the Lessonii group. SP86496; Q = 0.74 to the Lessonii group).

Removing the three northern Pseudopanax crassifolius samples that were sympatric with P. lessoniii plus all of the P. lessonii samples led to the recovery under TC-NJ of a monophyletic Crassifolius group (<50% TC-NJ BS), with P. crassifolius + chathamicus being sister to P. ferox (<50% TC-NJ BS), rather than to the Lessonii group which, after the removal of all P. lessonii samples, was represented by P. discolor and P. gilliesii. This suggested that introgression from P. lessonii into the northern P. crassifolius samples may have led to all of the P. crassifolius samples being ‘pulled’ toward the Lessonii group in the TC-NJ. However, the above alteration depended on the deletion of all three northern P. crassifolius samples, while the presence of a single P. lessonii sample was sufficient to ‘pull’ the remaining P. crassifolius + chathamicus samples towards the Lessonii group, making the Crassifolius group paraphyletic (or metaphyletic). The deletion of these samples had no effect on the NL-NJ result, and it also did not lead to the recovery of a monophyletic Crassifolius group in a repeat of the BA (where the remaining P. crassifolius + chathamicus samples formed a trichotomy with the Lessonii group and a clade of P. ferox plus P. linearis). The automated scoring produced a set with 621 characters, of which 605 were polymorphic and 519 were parsimony informative. The TC-distance error rate was slightly higher, with a median of 3.0% and a range of 1.1–6.6%. The tree-building results (not shown) were broadly similar to those from the manual scoring, consistent with the observation of Bonin et al. (2004) that signal within AFLP data can be robust to different scoring methods. The only difference of significance to the principal results presented above was that the Crassifolius group was no longer monophyletic in the MP, with Pseudopanax crassifolius and P. chathamicus being sister to the Lessonii group in all of the 200 MP trees, with 58% MP BS. Noteworthy results, that occurred across all tree-building methods albeit being poorly supported, were that P. crassifolius was monophyletic (74% NJ-TC, 68% NJ-NL, and <50% MP BS, 0.96 PP), rather than having P. chathamicus nested within it, and P. discolor and P. gilliesii were sister to one another (<50% NJ-TC, 65% NJNL, and <50% MP BS, 0.67 PP). 3.2. Chloroplast DNA sequences There were no significant results for the pairwise partition homogeneity tests, with p-values ranging from 0.47 to 1.0 for the nine-species set, and 0.98–0.99 for the 18-species set. For the nine-species set, the total alignment (Supplementary Nexus File 1) comprised 5916 bp, reducing to 5661 bp with ambiguous sites excluded (alignment positions: 222–224, 442, 538–540, 772–791, 1730–1770, 2641–2653, 5053–5094, 5579–5666, 5873–5916). The Pseudopanax crassifolius and P. lessonii samples differed by a single apomorphic substitution in the trnfM–trnS region of the former; additional sampling of P. crassifolius showed this was not a synapomorphy (see below). Meryta was sister to a monophyletic Pseudopanax in one of the two trees (score = 152) from MP without indel recoding (47% MP BS), the single tree (score = 182) from MP with indels recoded (57% MP BS), and in the BA (0.58 PP). ML BS for this relationship was 39%, and it was supported by a one bp insertion in the psbA–trnK spacer and one substitution in the trnfM–trnS region. Meryta was sister to the Arboreus group in the second tree from MP without indel recoding (48% MP BS; 23% MP BS with indels recoded; 47% ML BS; 0.34 PP; one substitution in the rps4 region). A 7 bp indel in the rps4 region supported Meryta as sister to the Crassifolius + Lessonii union, but this relationship had low BS and PP. The relationship between Meryta and Pseudopanax was unresolved in the single ML tree ( ln likelihood = 8802.74658). Pseudopanax crassifolius was more closely related to P. lessonii than to P. linearis in all of the analyses (100% MP and ML BS, 1.0 PP; five substitutions dispersed across four amplicons); a Shimodaira–Hasegawa test indicated that the topol-

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ogy constraining P. crassifolius and P. lessonii was significantly better under ML than that grouping P. crassifolius and P. linearis (p = 0.027). The alignment (Supplementary Nexus File 2) for the 18-species set comprised 3335 bp, reducing to 3290 bp with ambiguous sites excluded (alignment positions: 1–18, 910–915, 2043–2061, 3334– 3335). Two MP (score = 100; with no recoding of indels) trees, one of which is shown in Fig. 5, and a single ML tree ( ln likelihood = 5300.67143) were recovered. Monophyly of Meryta (both MP trees; 99% MP and 100% ML BS; 1.0 PP), the Arboreus group (both MP trees; 100% MP and 100% ML BS; 1.0 PP), and the Crassifolius + Lessonii union (both MP trees; 95% MP and 95% ML BS; 1.0 PP) was strongly supported. Within the Arboreus group, Pseudopanax laetus was supported as sister to the others (both MP trees; 86% MP and 90% ML BS; 1.0 PP), amongst which there was little resolution. All samples from the Lessonii group had identical concatenated haplotypes (other than an autapomorphic six bp indel in WELT SP86459) to two of the P. crassifolius samples, the P. chathamicus sample, and one of the P. ferox samples. The remaining P. crassifolius and P. ferox samples differed by one or two autapomorphic substitutions. Pseudopanax linearis was strongly supported as sister to the remaining samples of the Crassifolius + Lessonii union (both MP trees; 99% MP and 98% ML BS; 1.0 PP; four substitutions, with at least one in each of the three amplicons). Meryta was recovered as sister to the Arboreus group in one of the two MP trees (44% BS), and the ML (57% BS) and BA (0.58 PP) trees. Pseudopanax was monophyletic in the other MP tree (37% MP and 18% ML BS; 0.37 PP).

4. Discussion Other than the two species-pairs involving the island endemics, Pseudopanax arboreus–P. kermadecensis and P. crassifolius–P. chathamicus, AFLP DNA-fingerprinting clearly distinguishes the different

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Pseudopanax species. Indeed, most of the species are strongly supported as monophyletic, the exceptions being: P. arboreus which has P. kermadecensis nested within it; P. crassifolius which has P. chathamicus nested within it (with the manual scoring, and is only weakly supported as monophyletic with the automated scoring); and the heterogeneous P. colensoi (see Section 4.3).Some higher-level relationships seem robust, such as the Lessonii group, the Crassifolius + Lessonii union, and the Arboreus group; however, many are not, such as the relationships within the Arboreus and Lessonii groups, and the relationships of the Crassifolius group’s species to one another and to the Lessonii group. Although AFLP appears to be very good for distinguishing species, an inability to strongly resolve higher-level relationships appears to be a general property of phylogenetic investigations employing AFLP (e.g., Pelser et al., 2003; Perrie et al., 2003b; Tremetsberger et al., 2006; van den Berg and Groendijk-Wilders, 2007; Koopman et al., 2008; Meudt and Bayly, 2008). It is unclear if this is a fair reflection of these relationships (i.e., they are not strongly ‘tree-like’), or suggests a limitation of the method in this context. In Pseudopanax, it is interesting that it is the relationships at an intermediate level – amongst species within both of the two major groups – that are the least confidently resolved. 4.1. Generic taxonomy The Arboreus group and the union of the Crassifolius + Lessonii groups are clearly distinct in both the AFLP and chloroplast sequence data. However, these data do not improve the understanding of these groups’ relationships to one another or to other related Araliaceae, especially Meryta, and the Pacific Schefflera subgroup. This is for several reasons: samples of the latter were not available for this study; AFLP appears too variable to enable satisfactory outgroup rooting of this broader group, at least with the methodology used here; there is a lack of strong support from analyses of the chloroplast sequence data for any of the three possible relationships amongst the two Pseudopanax groups and Meryta; and, in any case, using sequence data from the chloroplast as a basis for taxonomic classification may be inappropriate if chloroplast introgression occurs in this group (see below). Thus, while there is no strong genetic evidence for the monophyly of Pseudopanax (circumscribed here as the Arboreus, Crassifolius, and Lessonii groups, and excluding species attributed to Raukaua and/or from outside New Zealand; Mitchell and Wagstaff, 2000), there is equally no convincing evidence that this circumscription is not monophyletic. Consequently, and in the interests of taxonomic stability, we see at present no compelling reason to re-segregate the Arboreus group as Neopanax, as has been done by Frodin and Govaerts (2004) and Tronchet et al. (2005). 4.2. Chloroplast discordance

Fig. 5. One of two MP trees of the chloroplast DNA sequence data for the 18-species set of Pseudopanax and outgroups. The numbers by the branches are MP BS, ML BS, and PP. See Table 1 for sample details.

The chloroplast sequences for all of the species in the Crassifolius + Lessonii union, except Pseudopanax linearis, are very similar (Fig. 5). Synapomorphies for either P. crassifolius or P. lessonii were not found amongst nearly 6000 bp of chloroplast sequence, meaning assessment of parental symmetry in the hybridisation between these two species is not yet possible. Plunkett et al. (2004) reported that their ITS and chloroplast sequence sets were generally congruent for relationships across the Araliaceae. However, with the chloroplast data presented here, the strongly supported (BS and Shimodaira–Hasegawa test) position of Pseudopanax linearis as sister to the remainder of the species in the Crassifolius + Lessonii union is inconsistent with both the ITS sequences of Mitchell and Wagstaff (1997) and our AFLP data. This conflict is not strong in that monophyly of the Crassifolius group in the ITS data is supported by only a single substitution, while the

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position of P. linearis is not confidently resolved by AFLP. Pseudopanax linearis is, nevertheless, never favoured as sister to the other species of the Crassifolius + Lessonii union in any of the tree-building analyses of the AFLP data. Furthermore, the large phenetic gap between P. linearis and the other species in the chloroplast data is not at all evident in the AFLP data. These anomalies, in both cladistic position and phenetic distance, are suggestive of chloroplast introgression. Although lineage-sorting cannot be completely discounted, it would require very unequal anagenetic change amongst the chloroplast lineages and/or ancestors polymorphic for rather different chloroplast lineages; the latter being at odds with the limited infra-specific polymorphisms presently observed. Additional clarification of this situation will require better resolution of the phylogeny for Pseudopanax with nuclear data, particularly the relationships of the Crassifolius group’s species, since the chloroplast phylogeny appears robust and includes multiple, geographically-spread samples of the key species (i.e., P. crassifolius, P. ferox, P. lessonii, and P. linearis). 4.3. The Arboreus group The taxonomic status of Pseudopanax colensoi requires further evaluation, with its infra-specific taxonomy (Wardle, 1968) not well-supported in the AFLP; in particular, var. ternatus was never monophyletic. Samples of P. colensoi var. fiordensis were consistently distinguished by AFLP from the other P. colensoi samples, although geographic sampling of the former would have to be extended before confident re-assessments could be made. Pseudopanax arboreus + kermadecensis, P. laetus, and P. macintyrei (see Wardle, 1968) are clearly distinguished in the AFLP, but the relationships between them are not. 4.4. The island endemics The Kermadec Islands’ endemic Pseudopanax kermadecensis and the Chatham Islands’ endemic P. chathamicus are both strongly recovered as monophyletic. At least with the present sampling, they are genetically less diverse than their respective mainland progenitors, P. arboreus and P. crassifolius. Multiple dispersal events to the Chatham Islands have been detected within other species (Shepherd et al., in press), but there is no evidence for substantial ongoing geneflow between mainland New Zealand and the offshore islands in Pseudopanax, and P. chathamicus and P. kermadecensis could well have each been founded by a single dispersal event. Furthermore, the nesting of Pseudopanax kermadecensis within P. arboreus means the former cannot be recognised as a distinct evolutionary lineage on the basis of its phylogenetic positioning in the AFLP data alone, since it appears equivalent to the subcomponents of the latter species. The situation with the Chatham Islands’ endemic P. chathamicus and P. crassifolius is similar, although there is some indication of reciprocal-monophyly in the automated AFLP scoring. Such nesting suggests that using similar methods to those here to investigate the taxonomic status of island populations whose morphological distinctiveness is less clear-cut (e.g., Perrie et al., 2003c) may be too conservative. Indeed, the relative rates of coalescence to reciprocal-monophyly with multiple, independent, neutral markers and the development of morphological differentiation and reproductive barriers requires further investigation (Coyne and Orr, 2004), especially in cases likely involving founding events; the island-endemic Pseudopanax species appeal as candidates for such study. 4.5. Pseudopanax crassifolius and hybridisation One of the most surprising results from the AFLP analyses was the absence of strong support for a monophyletic Crassifolius group.

Pseudopanax crassifolius (plus the related island-endemic P. chathamicus) variously grouped with P. ferox and P. linearis to form a monophyletic Crassifolius group, or with the Lessonii group; neither position was well-supported. Mitchell and Wagstaff (1997) did recover a monophyletic Crassifolius group. They listed three supporting morphological characters (with the character states from A. Mitchell, pers. comm., 2008): adult leaf type (simple), adult leaf primary venation (pinnate), and adult leaf texture (thick and coriaceous). However, these three characters are labile throughout the Araliaceae and, for at least the first two characters, the states found in Pseudopanax also occur elsewhere (Mitchell and Wagstaff, 1997; Plunkett et al., 2004). Furthermore, reappraisal of the ITS sequence data of Mitchell and Wagstaff (1997) indicates there is only a single synapomorphic substitution for the Crassifolius group. Thus, despite general intuition (including our own) that, because the species are similar morphologically, the Crassifolius group is ‘natural’, there is actually very little evidence for its monophyly. The inability to decisively establish the relationship of Pseudopanax crassifolius within the Crassifolius + Lessonii union means we cannot reconstruct the evolution of heteroblasty in the wider group. It also raises the possibility that hybridisation with P. lessonii may be influencing our ability to recover the ‘species’ phylogeny. This may be hinted at with the change to monophyly of the Crassifolius group in the TC-NJ when all of the P. lessonii samples and the northern P. crassifolius samples are excluded; these samples are the most likely to have experienced introgression given their sympatry. However, this effect does not occur with the NL-NJ or BA, and there is little indication of introgression between P. crassifolius and P. lessonii in the neighbour-net, which is capable of displaying conflicting phylogenetic signal, or in the Structure analysis, which is very good at detecting at least early generation hybrids in Pseudopanax (Perrie and Shepherd, unpubl. data). In any case, the northern P. crassifolius samples, which are broadly sympatric with P. lessonii, are more closely related to the remaining P. crassifolius samples, which are allopatric with P. lessonii, indicating any introgression is insufficient to disrupt the ‘cohesiveness’ of the two species. Nevertheless, the extent to which introgression can affect both the recovery of ‘species’ phylogenies and the phylogenies themselves when using multiple, independent markers (including AFLP) warrants further study. In hybridising groups, all genetic markers not causing or linked to the elements responsible for species’ differences might introgress. A general issue for hybridising groups is, therefore, whether phylogenies recovered from even multiple unlinked loci reflect the original order of speciation events, or subsequent introgression, and which particular loci are reflecting what? For instance, it is intriguing that in one of the most intensively studied examples of hybridisation, Helianthus annuus and H. petiolaris are resolved as relatively distantly related by nuclear ribosomal sequences (Timme et al., 2007), but exhibit extremely little genomic differentiation, and even less than that between H. annuus and its sister species from the ribosomal analyses, H. argophyllus (Yatabe et al., 2007). In Pseudopanax, if P. crassifolius is actually more closely related to the Lessonii group, it might explain why it hybridises much more frequently with P. lessonii than with either of the more morphologically-similar P. ferox and P. linearis with which it also frequently co-occurs. However, it is unclear how we could readily distinguish the hypothesis that P. crassifolius and P. lessonii hybridise because they are related from the hypothesis that they appear related because they hybridise. Whatever their precise relationship, and despite their frequent and widespread hybridisation, Pseudopanax crassifolius and P. lessonii are clearly resolved in the AFLP as separate, non-sister evolutionary lineages, at least when only apparently ‘pure’ individuals are investigated. This provides a framework for further investigation of their hybridisation: What do F1’s look like, and how common are they? To what extent does hybridisation proceed

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beyond F1’s, and is there evidence for local introgression? What are the significant barriers to gene-flow, and why are the parental species so clearly resolved when they hybridise so commonly? Addressing these issues in these southern hemisphere trees will broaden understanding of hybridisation and, in turn, the factors important as barriers to gene-flow between species. Acknowledgments This work was primarily funded by the New Zealand Marsden Fund (contract MNZ0501), with additional contributions from New Zealand’s Royal Forest and Bird Protection Society’s J.S. Watson Conservation Trust and the Wellington Botanical Society’s Jubilee Award. We thank the Auckland Regional Council, Department of Conservation, Otari-Wilton’s Bush, Peter de Lange, Hilary Donald, David Havell, Cameron Hay, Peter Heenan, Rodney Lewington, Anthony Mitchell, Barbara Parris, Alton Perrie, Barry Sneddon, and Mike Shepherd for their assistance with obtaining samples; Helen Mechen and Jaime Dörner for processing herbarium specimens; and Anthony Mitchell, Rob Smissen, and two anonymous reviewers for comments on the draft manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev.2009.05.030. References Albach, D.C., 2007. Amplified fragment length polymorphisms and sequence data in the phylogenetic analysis of polyploids: multiple origins of Veronica cymbalaria (Plantaginaceae). New Phytologist 176, 481–498. Arnold, J., 1993. Cytonuclear disequilibria in hybrid zones. Annual Review of Ecology and Systematics 24, 521–554. Bonin, A., Bellemain, E., Eidesen, P.B., Pompanon, F., Brochmann, C., Taberlet, P., 2004. How to track and assess genotyping errors in population genetics studies. Molecular Ecology 13, 3261–3273. Burns, K.C., 2005. Plastic heteroblasty in beach grondsel (Senecio lautus). New Zealand Journal of Botany 43, 665–672. Burns, K.C., Dawson, J.W., 2006. A morphological comparison of leaf heteroblasty between New Caledonia and New Zealand. New Zealand Journal of Botany 44, 387–396. Cockanye, L., Allan, H.H., 1926. Notes on New Zealand floristic botany, including descriptions of new species, &c. (No. 4). Transactions of the New Zealand Institute 56, 21–33. Coyne, J.A., Orr, H.A., 2004. Speciation. Sinauer Associates, Sunderland. Doyle, J.J., Doyle, J.D., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13– 15. Falush, D., Stephens, M., Pritchard, J.K., 2007. Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Notes 7, 574–578. Farris, J.S., Källersjö, M., Kluge, A.G., Bull, C., 1994. Testing significance of incongruence. Cladistics 10, 315–319. Frodin, D.G., Govaerts, R., 2004. World Checklist and Bibliography of Araliaceae. Kew Publishing, London. Harrison, R.G., 1993. Hybrids and hybrid zones: historical perspective. In: Harrison, R.G. (Ed.), Hybrid Zones and the Evolutionary Process. Oxford University Press, Oxford, pp. 3–12. Harrison, R.G., 1998. Linking evolutionary pattern and process: the relevance of species concepts for the study of speciation. In: Howard, D.J., Berlocher, S.H. (Eds.), Endless Forms. Species and Speciation. Oxford University Press, New York, pp. 19–31. Hey, J., 2006. Recent advances in assessing gene flow between diverging populations and species. Current Opinion in Genetics & Development 16, 592–596. Huelsenbeck, J.P., Ronquist, F.R., 2001. MrBayes: Bayesian inference of phylogeny. Biometrics 17, 754–755. Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies. Molecular Biology and Evolution 23, 254–267. Koopman, W.J.M., 2005. Phylogenetic signal in AFLP data sets. Systematic Biology 54, 197–217. Koopman, W.J.M., Wissemann, V., De Cock, K., Van Huylenbroeck, J.V., De Riek, J., Sabatino, G.J.H., Visser, D., Vosman, B., Ritz, C.M., Maes, B., Werlemark, G., Nybom, H., Debener, T., Linde, M., Smulders, M.J.M., 2008. AFLP markers as a tool to reconstruct complex relationships: a case study in Rosa (Rosaceae). American Journal of Botany 95, 353–366. Lexer, C., Fay, M.F., Joseph, J.A., Nica, M.-S., Heinze, B., 2005. Barriers to gene flow between two ecologically divergent Populus species, P. Alba (white poplar) and

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